Vaccines for human papilloma virus and methods for using the same

ABSTRACT

Improved anti-HPV immunogens and nucleic acid molecules that encode them are disclosed. Immunogens disclosed include those having consensus HPV6 E6E7, HPV11 E6E7, HPV16 E6E7, HPV18 E6E7, HPV31 E6E7, HPV33 E6E7, HPV39 E6E7, HPV45 E6E7, HPV52 E6E7, and HPV58 E6E7. Pharmaceutical composition, recombinant vaccines comprising DNA plasmid and live attenuated vaccines are disclosed as well methods of inducing an immune response in an individual against HPV are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national stage application filed under 35U.S.C. § 371 claiming benefit to International Patent Application No.PCT/US14/025106, filed Mar. 12, 2014, which is entitled to priorityunder 35 U.S.C. § 119(e) to U.S. Provisional Application No. 61/777,198,filed Mar. 12, 2013, each of which is hereby incorporated by referencein its entirety.

FIELD OF THE INVENTION

The present invention relates to improved human papillomavirus (HPV)vaccines, improved methods for inducing immune responses, and forprophylactically and/or therapeutically immunizing individuals againstHPV.

BACKGROUND OF THE INVENTION

Papillomavirus are small DNA viruses that comprise up to seven earlygenes and two late genes. Generally, papilloma virus early genes aredesignated E1-E7, and papilloma virus late genes are designated L1 andL2. Several species of animals can be infected by members of thepapillomavirus family.

Human Papillomavirus (HPV) infection is common and can be transmittedsexually. HPV have been differentiated into 56 or more types based uponDNA sequence homology. HPV types 16 and 18, which cause epithelialdysplasia and other lesions, are often associated with an increased riskof cancer, particularly in situ and invasive carcinomas of the cervix,vagina, vulva and anal canal. Nearly 88% of cervical cancers worldwideare the result of HPV subtypes 16, 18, 45, 31, 33, 52 and 58.Furthermore, various studies have revealed the presence of HPV6 andHPV11 in most incidences of recurrent respiratory papillomatosis. Thoughknown for their association with genital warts and only found in a smallpercentage of cervical cancer cases, HPV6 and HPV11 have been found in2.6-5.2% of all cases of low-grade cervical lesions. Furthermore, HPV6and HPV11 have now been associated with approximately 20% of low-gradesquamous intraepithelial lesions that are now considered precursors tocervical cancer, including grade 2 and 3 cervical intraepithelialneoplasia and mild cervical dysplasia. Increasing studies have revealedthe association of the two serotypes with various forms ofotolaryngologic diseases, including genital warts, recurrent respiratorypapillomatosis, lung carcinoma, tonsillar carcinoma, laryngealcarcinoma, and other malignant transformations of otherwise benignneoplasms and dysplasia of the head and neck. The findings of this studyshed light and future promise in the use of consensus sequence DNAvaccines against HPV6 and HPV11.

DNA vaccines have many conceptual advantages over more traditionalvaccination methods, such as live attenuated viruses and recombinantprotein-based vaccines. DNA vaccines are safe, stable, easily produced,and well tolerated in humans with preclinical trials indicating littleevidence of plasmid integration [Martin, T., et al., Plasmid DNA malariavaccine: the potential for genomic integration after intramuscularinjection. Hum Gene Ther, 1999. 10(5): p. 759-68; Nichols, W. W., etal., Potential DNA vaccine integration into host cell genome. Ann NYAcad Sci, 1995. 772: p. 30-9]. In addition, DNA vaccines are well suitedfor repeated administration due to the fact that efficacy of the vaccineis not influenced by pre-existing antibody titers to the vector[Chattergoon, M., J. Boyer, and D. B. Weiner, Genetic immunization: anew era in vaccines and immune therapeutics. FASEB J, 1997. 11(10): p.753-63]. However, one major obstacle for the clinical adoption of DNAvaccines has been a decrease in the platforms immunogenicity when movingto larger animals [Liu, M. A. and J. B. Ulmer, Human clinical trials ofplasmid DNA vaccines. Adv Genet, 2005. 55: p. 25-40]. Recenttechnological advances in the engineering of DNA vaccine immunogen, suchhas codon optimization, RNA optimization and the addition ofimmunoglobulin leader sequences have improved expression andimmunogenicity of DNA vaccines [Andre, S., et al., Increased immuneresponse elicited by DNA vaccination with a synthetic gp120 sequencewith optimized codon usage. J Virol, 1998. 72(2): p. 1497-503; Deml, L.,et al., Multiple effects of codon usage optimization on expression andimmunogenicity of DNA candidate vaccines encoding the humanimmunodeficiency virus type 1 Gag protein. J Virol, 2001. 75(22): p.10991-1001; Laddy, D. J., et al., Immunogenicity of novelconsensus-based DNA vaccines against avian influenza. Vaccine, 2007.25(16): p. 2984-9; Frelin, L., et al., Codon optimization and mRNAamplification effectively enhances the immunogenicity of the hepatitis Cvirus nonstructural 3/4A gene. Gene Ther, 2004. 11(6): p. 522-33], aswell as, recently developed technology in plasmid delivery systems suchas electroporation [Hirao, L. A., et al., Intradermal/subcutaneousimmunization by electroporation improves plasmid vaccine delivery andpotency in pigs and rhesus macaques. Vaccine, 2008. 26(3): p. 440-8;Luckay, A., et al., Effect of plasmid DNA vaccine design and in vivoelectroporation on the resulting vaccine-specific immune responses inrhesus macaques. J Virol, 2007. 81(10): p. 5257-69; Ahlen, G., et al.,In vivo electroporation enhances the immunogenicity of hepatitis C virusnonstructural 3/4A DNA by increased local DNA uptake, proteinexpression, inflammation, and infiltration of CD3+ T cells. J Immunol,2007. 179(7): p. 4741-53]. In addition, studies have suggested that theuse of consensus immunogens may be able to increase the breadth of thecellular immune response as compared to native antigens alone [Yan., J.,et al., Enhanced cellular immune responses elicited by an engineeredHIV-1 subtype B consensus-based envelope DNA vaccine. Mol Ther, 2007.15(2): p. 411-21; Rolland, M., et al., Reconstruction and function ofancestral center-of-tree human immunodeficiency virus type 1 proteins. JVirol, 2007. 81(16): p. 8507-14].

There remains a need for improved vaccines and methods for preventingand treating HPV infection, in particular infections leading to cervicalcancer and/or carcinomas of the lung, tonsil, and larynx.

SUMMARY OF THE INVENTION

Aspects of the invention provide compositions comprising at least onenucleotide sequence comprising an HPV E6-E7 fusion antigen selected fromthe group consisting of: HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV39,HPV45, HPV52, and HPV58.

Another aspect provides compositions comprising one or more nucleotidesequences encoding an HPV E6-E7 fusion antigen selected from the groupconsisting of: nucleotide sequence that encodes SEQ ID NO:2; nucleotidesequence that encodes SEQ ID NO:4; nucleotide sequence that encodes SEQID NO:6; nucleotide sequence that encodes SEQ ID NO:8; nucleotidesequence that encodes SEQ ID NO:18; nucleotide sequence that encodes SEQID NO:20; nucleotide sequence that encodes SEQ ID NO:22; nucleotidesequence that encodes SEQ ID NO:24; a nucleic acid sequence that is atleast 95% homologous to a nucleic acid sequence nucleotide sequence thatencodes SEQ ID NO:2; a nucleic acid sequence that is at least 95%homologous to a nucleotide sequence that encodes SEQ ID NO:4; a nucleicacid sequence that is at least 95% homologous to a nucleotide sequencethat encodes SEQ ID NO:6; a nucleic acid sequence that is at least 95%homologous to a nucleotide sequence that encodes SEQ ID NO:8; a nucleicacid sequence that is at least 95% homologous to a nucleotide sequencethat encodes SEQ ID NO:18; a nucleic acid sequence that is at least 95%homologous to a nucleotide sequence that encodes SEQ ID NO:20; a nucleicacid sequence that is at least 95% homologous to a nucleotide sequencethat encodes SEQ ID NO:22; a nucleic acid sequence that is at least 95%homologous to a nucleotide sequence that encodes SEQ ID NO:24; afragment of a nucleotide sequence that encodes SEQ ID NO:2; a fragmentof a nucleotide sequence that encodes SEQ ID NO:4; a fragment of anucleotide sequence that encodes SEQ ID NO:6; a fragment of a nucleotidesequence that encodes SEQ ID NO:8; a fragment of a nucleotide sequencethat encodes SEQ ID NO:18; a fragment of a nucleotide sequence thatencodes SEQ ID NO:20; a fragment of a nucleotide sequence that encodesSEQ ID NO:22; a fragment of a nucleotide sequence that encodes SEQ IDNO:24; a nucleic acid sequence that is at least 95% homologous to afragment of a nucleotide sequence that encodes SEQ ID NO:2; a nucleicacid sequence that is at least 95% homologous to a fragment of anucleotide sequence that encodes SEQ ID NO:4; a nucleic acid sequencethat is at least 95% homologous to a fragment of a nucleotide sequencethat encodes SEQ ID NO:6; a nucleic acid sequence that is at least 95%homologous to a fragment of a nucleotide sequence that encodes SEQ IDNO:8; a nucleic acid sequence that is at least 95% homologous to afragment of a nucleotide sequence that encodes SEQ ID NO:18; a nucleicacid sequence that is at least 95% homologous to a fragment of anucleotide sequence that encodes SEQ ID NO:20; a nucleic acid sequencethat is at least 95% homologous to a fragment of a nucleotide sequencethat encodes SEQ ID NO:22; and a nucleic acid sequence that is at least95% homologous to a fragment of a nucleotide sequence that encodes SEQID NO:24. In some embodiments, the nucleotide sequences encoding the HPVE6-E7 fusion antigen are without a leader sequence at 5′ end that is anucleotide sequence that encodes SEQ ID NO:10.

In another aspect of the invention, there are provided compositionscomprising one or more nucleotide sequences encoding an HPV E6-E7 fusionantigen selected from the group consisting of: SEQ ID NO:1; SEQ ID NO:3;SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQID NO:23; a nucleic acid sequence that is at least 95% homologous to SEQID NO:1; a nucleic acid sequence that is at least 95% homologous to SEQID NO:3; a nucleic acid sequence that is at least 95% homologous to SEQID NO:5; a nucleic acid sequence that is at least 95% homologous to SEQID NO:7; a nucleic acid sequence that is at least 95% homologous to SEQID NO:17; a nucleic acid sequence that is at least 95% homologous to SEQID NO:19; a nucleic acid sequence that is at least 95% homologous to SEQID NO:21; a nucleic acid sequence that is at least 95% homologous to SEQID NO:23; a fragment of SEQ ID NO:1; a fragment of SEQ ID NO:3; afragment of SEQ ID NO:5; a fragment of SEQ ID NO:7; a fragment of SEQ IDNO:17; a fragment of SEQ ID NO:19; a fragment of SEQ ID NO:21; afragment of SEQ ID NO:23; a nucleic acid sequence that is at least 95%homologous to a fragment of SEQ ID NO:1; a nucleic acid sequence that isat least 95% homologous to a fragment of SEQ ID NO:3; a nucleic acidsequence that is at least 95% homologous to a fragment of SEQ ID NO:5; anucleic acid sequence that is at least 95% homologous to a fragment ofSEQ ID NO:7; a nucleic acid sequence that is at least 95% homologous toa fragment of SEQ ID NO:17; a nucleic acid sequence that is at least 95%homologous to a fragment of SEQ ID NO:19; a nucleic acid sequence thatis at least 95% homologous to a fragment of SEQ ID NO:21; and a nucleicacid sequence that is at least 95% homologous to a fragment of SEQ IDNO:23. In some embodiments, the nucleotide sequences encoding the HPVE6-E7 fusion antigen are without a leader sequence at 5′ end that hasnucleotide sequence SEQ ID NO:9.

The nucleotide sequences provided can be a plasmid.

In additional aspects, provided are pharmaceutical compositionscomprising the disclosed nucleotide sequences; preferably with multipleanitgens.

In some aspects, there are methods of inducing an effective immuneresponse in an individual against more than one subtype of HPV,comprising administering to said individual a composition comprising oneor more of the nucelotides sequences provided; preferably, thecompositions have more than one antigen. The methods preferably includea step of introducing the provided nucleotide sequences into theindividual by electroporation.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1. Phylogenetic trees based on neighbor-joining evaluation of E6and E7 alignments (A and B, respectively). Asterisks indicate locationof consensus sequences on each tree.

FIG. 2. In vivo expression of p6E6E7 and p11E6E7. Gene products wereisolated from lysed transfected 293-T cells, run through SDS-PAGE gel,and detected using autoradiography. Both HPV 6 and HPV 11 E6/E7 proteinsare approximately 32 kDa each (A). Human rhabdomyosarcoma (RD) cellswere also transfected with p6E6E7 and p11E6E7 and later fixed afterimmunofluorescence staining. FITC fluorescence confirms expression ofp6E6E7 and p11E6E7(B). DAPI fluorescence confirms nuclei localizationconsequent of Hoescht staining.

FIG. 3. IFN-γ ELISpot assays show induction of robust cell-mediatedresponses by p6E6E7 (A) and p11E6E7 (B) in C57BL/6 mice. Assays wereperformed using splenocytes isolated from mice in each respective group(5 mice per group) after three biweekly immunizations. Each immunizationconsisted of 20 μg per construct. Mice in combo group received bothp6E6E7 and p11E6E7 at 20 μg per construct, for a total of 40 μg DNA perimmunization. DNA was administered via IM injection, followed byelectroporation (* denotes p<0.0001; † denotes p=0.0001).

FIG. 4. Additional IFN-γ ELISpot assays performed using individualpeptides to characterize dominant epitopes. Splenocytes isolated fromvaccinated mice and negative control were stimulated with overlappingpeptides that span the entire HPV 6 E6/E7 fusion protein (A) or HPV 11E6/E7 fusion protein (B).

FIG. 5. Cytokine production by antigen-specific T-cells characterized byintracellular cytokine staining. Splenocytes isolated from micevaccinated with p6E6E7 (A) and p11E6E7 (B) were stimulated with R10growth medium, PMA, or consensus gene peptides for 4 hours prior tosurface marker and intracellular staining. Dot plots above showdifferences in background-subtracted percentages of either total CD4+ orCD8+ cells producing IFN-γ, IL-2, and TNF-α. P-values for plots withasterisks were unable to be determined because the average of thenegative control group was zero.

FIG. 6. Displays the amino acid sequence for HPV31 E6/E7 (SEQ ID NO:18)annotated to show the IgE leader sequence (IgEL), the endoproteolyticcleavage site, and sites where the E6 and E7 domain were mutated toenhance expression and immunogenicity.

FIG. 7. Displays the amino acid sequence for HPV52 E6/E7 (SEQ ID NO:20)annotated to show the IgE leader sequence (IgEL), the endoproteolyticcleavage site, and sites where the E6 and E7 domain were mutated toenhance expression and immunogenicity.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Definitions

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used in thespecification and the appended claims, the singular forms “a,” “an” and“the” include plural referents unless the context clearly dictatesotherwise.

For recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated.For example, for the range of 6-9, the numbers 7 and 8 are contemplatedin addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitlycontemplated.

a. Adjuvant

“Adjuvant” as used herein may mean any molecule added to the DNA plasmidvaccines described herein to enhance antigenicity of the one or moreantigens encoded by the DNA plasmids and encoding nucleic acid sequencesdescribed hereinafter.

b. Antibody

“Antibody” may mean an antibody of classes IgG, IgM, IgA, IgD or IgE, orfragments, fragments or derivatives thereof, including Fab, F(ab′)2, Fd,and single chain antibodies, diabodies, bispecific antibodies,bifunctional antibodies and derivatives thereof. The antibody may be anantibody isolated from the serum sample of mammal, a polyclonalantibody, affinity purified antibody, or mixtures thereof which exhibitssufficient binding specificity to a desired epitope or a sequencederived therefrom.

c. Antigen

“Antigen” refers to: proteins having an HPV E6 or HPV E7 domain, andpreferably and E6 and E7 fusion with an endeoproteolytic cleavage sitetherebetween. Antigens include SEQ ID NOs: 2 (subtype 6), 4 (subtype11), 6 (subtype 33), 8 (subtype 58), 18 (subtype 31), 20 (subtype 52),22 (subtype 16), and 24 (subtype 18); fragments thereof of lengths setforth herein, variants, i.e. proteins with sequences homologous to SEQID NOs:2, 4, 6, 8, 18, 20, 22, and 24 as set forth herein, fragments ofvariants having lengths set forth herein, and combinations thereofAnitgens may have an IgE leader sequence of SEQ ID NO:10 or mayalternatively have such sequence removed from the N-terminal end.Antigens may optionally include signal peptides such as those from otherproteins.

d. Coding Sequence

“Coding sequence” or “encoding nucleic acid” as used herein may meanrefers to the nucleic acid (RNA or DNA molecule) that comprise anucleotide sequence which encodes an antigen as set forth in section c.above. The coding sequence may further include initiation andtermination signals operably linked to regulatory elements including apromoter and polyadenylation signal capable of directing expression inthe cells of an individual or mammal to whom the nucleic acid isadministered. The coding sequence may further include sequences thatencode signal peptides, e.g., an IgE leader sequence such as SEQ IDNO:9.

e. Complement

“Complement” or “complementary” as used herein may mean a nucleic acidmay mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairingbetween nucleotides or nucleotide analogs of nucleic acid molecules.

f. Fragment

“Fragment” may mean a polypeptide fragment of an antigen that is capableof eliciting an immune response in a mammal against the antigen. Afragment of an antigen may be 100% identical to the full length exceptmissing at least one amino acid from the N and/or C terminal, in eachcase with or without signal peptides and/or a methionine at position 1.Fragments may comprise 60% or more, 65% or more, 70% or more, 75% ormore, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more,93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% ormore, 99% or more percent of the length of the particular full lengthantigen, excluding any heterologous signal peptide added. The fragmentmay, preferably, comprise a fragment of a polypeptide that is 95% ormore, 96% or more, 97% or more, 98% or more or 99% or more homologous tothe antigen and additionally comprise an N terminal methionine orheterologous signal peptide which is not included when calculatingpercent homology Fragments may further comprise an N terminal methionineand/or a signal peptide such as an immunoglobulin signal peptide, forexample an IgE or IgG signal peptide. The N terminal methionine and/orsignal peptide may be linked to a fragment of an antigen.

A fragment of a nucleic acid sequence that encodes antigen may be 100%identical to the full length except missing at least one nucleotide fromthe 5′ and/or 3′ end, in each case with or without sequences encodingsignal peptides and/or a methionine at position 1. Fragments maycomprise 60% or more, 65% or more, 70% or more, 75% or more, 80% ormore, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more,94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% ormore percent of the length of the particular full length codingsequence, excluding any heterologous signal peptide added. The fragmentmay, preferably, comprise a fragment that encodes a polypeptide that is95% or more, 96% or more, 97% or more, 98% or more or 99% or morehomologous to the antigen and additionally optionally comprise sequenceencoding an N terminal methionine or heterologous signal peptide whichis not included when calculating percent homology Fragments may furthercomprise coding sequences for an N terminal methionine and/or a signalpeptide such as an immunoglobulin signal peptide, for example an IgE orIgG signal peptide. The coding sequence encoding the N terminalmethionine and/or signal peptide may be linked to a fragment of codingsequence.

g. Identical

“Identical” or “identity” as used herein in the context of two or morenucleic acids or polypeptide sequences, may mean that the sequences havea specified percentage of residues that are the same over a specifiedregion. The percentage may be calculated by optimally aligning the twosequences, comparing the two sequences over the specified region,determining the number of positions at which the identical residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the specified region, and multiplying the result by 100 toyield the percentage of sequence identity. In cases where the twosequences are of different lengths or the alignment produces one or morestaggered ends and the specified region of comparison includes only asingle sequence, the residues of single sequence are included in thedenominator but not the numerator of the calculation. When comparing DNAand RNA, thymine (T) and uracil (U) may be considered equivalent.Identity may be performed manually or by using a computer sequencealgorithm such as BLAST or BLAST 2.0.

h. Immune Response

“Immune response” as used herein may mean the activation of a host'simmune system, e.g., that of a mammal, in response to the introductionof one or more antigens via the provided DNA plasmid vaccines. Theimmune response can be in the form of a cellular or humoral response, orboth.

i. Nucleic Acid

“Nucleic acid” or “oligonucleotide” or “polynucleotide” as used hereinmay mean at least two nucleotides covalently linked together. Thedepiction of a single strand also defines the sequence of thecomplementary strand. Thus, a nucleic acid also encompasses thecomplementary strand of a depicted single strand. Many variants of anucleic acid may be used for the same purpose as a given nucleic acid.Thus, a nucleic acid also encompasses substantially identical nucleicacids and complements thereof. A single strand provides a probe that mayhybridize to a target sequence under stringent hybridization conditions.Thus, a nucleic acid also encompasses a probe that hybridizes understringent hybridization conditions.

Nucleic acids may be single stranded or double stranded, or may containportions of both double stranded and single stranded sequence. Thenucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, wherethe nucleic acid may contain combinations of deoxyribo- andribo-nucleotides, and combinations of bases including uracil, adenine,thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosineand isoguanine. Nucleic acids may be obtained by chemical synthesismethods or by recombinant methods.

j. Operably Linked

“Operably linked” as used herein may mean that expression of a gene isunder the control of a promoter with which it is spatially connected. Apromoter may be positioned 5′ (upstream) or 3′ (downstream) of a geneunder its control. The distance between the promoter and a gene may beapproximately the same as the distance between that promoter and thegene it controls in the gene from which the promoter is derived. As isknown in the art, variation in this distance may be accommodated withoutloss of promoter function.

k. Promoter

“Promoter” as used herein may mean a synthetic or naturally-derivedmolecule which is capable of conferring, activating or enhancingexpression of a nucleic acid in a cell. A promoter may comprise one ormore specific transcriptional regulatory sequences to further enhanceexpression and/or to alter the spatial expression and/or temporalexpression of same. A promoter may also comprise distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A promoter may bederived from sources including viral, bacterial, fungal, plants,insects, and animals. A promoter may regulate the expression of a genecomponent constitutively, or differentially with respect to cell, thetissue or organ in which expression occurs or, with respect to thedevelopmental stage at which expression occurs, or in response toexternal stimuli such as physiological stresses, pathogens, metal ions,or inducing agents. Representative examples of promoters include thebacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lacoperator-promoter, tac promoter, SV40 late promoter, SV40 earlypromoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40late promoter and the CMV IE promoter.

l. Stringent Hybridization Conditions

“Stringent hybridization conditions” as used herein may mean conditionsunder which a first nucleic acid sequence (e.g., probe) will hybridizeto a second nucleic acid sequence (e.g., target), such as in a complexmixture of nucleic acids. Stringent conditions are sequence-dependentand will be different in different circumstances. Stringent conditionsmay be selected to be about 5 10° C. lower than the thermal meltingpoint (Tm) for the specific sequence at a defined ionic strength pH. TheTm may be the temperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at Tm, 50% of the probes are occupied atequilibrium). Stringent conditions may be those in which the saltconcentration is less than about 1.0 M sodium ion, such as about0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3and the temperature is at least about 30° C. for short probes (e.g.,about 10-50 nucleotides) and at least about 60° C. for long probes(e.g., greater than about 50 nucleotides). Stringent conditions may alsobe achieved with the addition of destabilizing agents such as formamide.For selective or specific hybridization, a positive signal may be atleast 2 to 10 times background hybridization. Exemplary stringenthybridization conditions include the following: 50% formamide, 5×SSC,and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65°C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.

m. Substantially Complementary

“Substantially complementary” as used herein may mean that a firstsequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or99% identical to the complement of a second sequence over a region of 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or morenucleotides or amino acids, or that the two sequences hybridize understringent hybridization conditions.

n. Substantially Identical

“Substantially identical” as used herein may mean that a first andsecond sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 98% or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100 or more nucleotides or amino acids, or withrespect to nucleic acids, if the first sequence is substantiallycomplementary to the complement of the second sequence.

o. Variant

“Variant” used herein with respect to a nucleic acid may mean (i) aportion or fragment of a referenced nucleotide sequence; (ii) thecomplement of a referenced nucleotide sequence or portion thereof; (iii)a nucleic acid that is substantially identical to a referenced nucleicacid or the complement thereof; or (iv) a nucleic acid that hybridizesunder stringent conditions to the referenced nucleic acid, complementthereof, or a sequences substantially identical thereto.

“Variant” with respect to a peptide or polypeptide that differs in aminoacid sequence by the insertion, deletion, or conservative substitutionof amino acids, but retain at least one biological activity. Variant mayalso mean a protein with an amino acid sequence that is substantiallyidentical to a referenced protein with an amino acid sequence thatretains at least one biological activity. A conservative substitution ofan amino acid, i.e., replacing an amino acid with a different amino acidof similar properties (e.g., hydrophilicity, degree and distribution ofcharged regions) is recognized in the art as typically involving a minorchange. These minor changes can be identified, in part, by consideringthe hydropathic index of amino acids, as understood in the art. Kyte etal., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an aminoacid is based on a consideration of its hydrophobicity and charge. It isknown in the art that amino acids of similar hydropathic indexes can besubstituted and still retain protein function. In one aspect, aminoacids having hydropathic indexes of ±2 are substituted. Thehydrophilicity of amino acids can also be used to reveal substitutionsthat would result in proteins retaining biological function. Aconsideration of the hydrophilicity of amino acids in the context of apeptide permits calculation of the greatest local average hydrophilicityof that peptide, a useful measure that has been reported to correlatewell with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101,incorporated fully herein by reference. Substitution of amino acidshaving similar hydrophilicity values can result in peptides retainingbiological activity, for example immunogenicity, as is understood in theart. Substitutions may be performed with amino acids havinghydrophilicity values within ±2 of each other. Both the hyrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties.

p. Vector

“Vector” used herein may mean a nucleic acid sequence containing anorigin of replication. A vector may be a plasmid, bacteriophage,bacterial artificial chromosome or yeast artificial chromosome. A vectormay be a DNA or RNA vector. A vector may be either a self-replicatingextrachromosomal vector or a vector which integrates into a host genome.

Improved vaccines are disclosed which arise from a multi-phase strategyto enhance cellular immune responses induced by immunogens. Modifiedconsensus sequences were generated. Genetic modifications includingcodon optimization, RNA optimization, and the addition of a highefficient immunoglobin leader sequence are also disclosed. The novelconstruct has been designed to elicit stronger and broader cellularimmune responses than a corresponding codon optimized immunogens.

The improved HPV vaccines are based upon proteins and genetic constructsthat encode proteins with epitopes that make them particularly effectiveas immunogens against which anti-HPV can be induced. Accordingly,vaccines may induce a therapeutic or prophylactic immune response. Insome embodiments, the means to deliver the immunogen is a DNA vaccine, arecombinant vaccine, a protein subunit vaccine, a composition comprisingthe immunogen, an attenuated vaccine or a killed vaccine. In someembodiments, the vaccine comprises a combination selected from thegroups consisting of: one or more DNA vaccines, one or more recombinantvaccines, one or more protein subunit vaccines, one or more compositionscomprising the immunogen, one or more attenuated vaccines and one ormore killed vaccines.

According to some embodiments, a vaccine is delivered to an individualto modulate the activity of the individual's immune system and therebyenhance the immune response against HPV. When a nucleic acid moleculethat encodes the protein is taken up by cells of the individual thenucleotide sequence is expressed in the cells and the protein arethereby delivered to the individual. Methods of delivering the codingsequences of the protein on nucleic acid molecule such as plasmid, aspart of recombinant vaccines and as part of attenuated vaccines, asisolated proteins or proteins part of a vector are provided.

Compositions and methods are provided which prophylactically and/ortherapeutically immunize an individual against HPV.

Compositions for delivering nucleic acid molecules that comprise anucleotide sequence that encodes the immunogen are operably linked toregulatory elements. Compositions may include a plasmid that encodes theimmunogen, a recombinant vaccine comprising a nucleotide sequence thatencodes the immunogen, a live attenuated pathogen that encodes a proteinof the invention and/or includes a protein of the invention; a killedpathogen includes a protein of the invention; or a composition such as aliposome or subunit vaccine that comprises a protein of the invention.The present invention further relates to injectable pharmaceuticalcompositions that comprise compositions.

Aspects of the invention provide compositions comprising at least onenucleotide sequence comprising an HPV E6-E7 fusion antigen selected fromthe group consisting of: HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV39,HPV45, HPV52, and HPV58. In some embodiments the compositions cancomprise HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV52, and HPV58. Insome embodiments the compositions can comprise HPV16, HPV18, HPV31,HPV33, HPV52, and HPV58. In some embodiments the compositions cancomprise HPV6, HPV11, HPV16, and HPV18. In some embodiments thecompositions can comprise HPV16, HPV31, and HPV52.

Another aspect provides compositions comprising one or more nucleotidesequences encoding an HPV E6-E7 fusion antigen selected from the groupconsisting of: nucleotide sequence that encodes SEQ ID NO:2; nucleotidesequence that encodes SEQ ID NO:4; nucleotide sequence that encodes SEQID NO:6; nucleotide sequence that encodes SEQ ID NO:8; nucleotidesequence that encodes SEQ ID NO:18; nucleotide sequence that encodes SEQID NO:20; nucleotide sequence that encodes SEQ ID NO:22; nucleotidesequence that encodes SEQ ID NO:24; a nucleic acid sequence that is atleast 95% homologous to a nucleic acid sequence nucleotide sequence thatencodes SEQ ID NO:2; a nucleic acid sequence that is at least 95%homologous to a nucleotide sequence that encodes SEQ ID NO:4; a nucleicacid sequence that is at least 95% homologous to a nucleotide sequencethat encodes SEQ ID NO:6; a nucleic acid sequence that is at least 95%homologous to a nucleotide sequence that encodes SEQ ID NO:8; a nucleicacid sequence that is at least 95% homologous to a nucleotide sequencethat encodes SEQ ID NO:18; a nucleic acid sequence that is at least 95%homologous to a nucleotide sequence that encodes SEQ ID NO:20; a nucleicacid sequence that is at least 95% homologous to a nucleotide sequencethat encodes SEQ ID NO:22; a nucleic acid sequence that is at least 95%homologous to a nucleotide sequence that encodes SEQ ID NO:24; afragment of a nucleotide sequence that encodes SEQ ID NO:2; a fragmentof a nucleotide sequence that encodes SEQ ID NO:4; a fragment of anucleotide sequence that encodes SEQ ID NO:6; a fragment of a nucleotidesequence that encodes SEQ ID NO:8; a fragment of a nucleotide sequencethat encodes SEQ ID NO:18; a fragment of a nucleotide sequence thatencodes SEQ ID NO:20; a fragment of a nucleotide sequence that encodesSEQ ID NO:22; a fragment of a nucleotide sequence that encodes SEQ IDNO:24; a nucleic acid sequence that is at least 95% homologous to afragment of a nucleotide sequence that encodes SEQ ID NO:2; a nucleicacid sequence that is at least 95% homologous to a fragment of anucleotide sequence that encodes SEQ ID NO:4; a nucleic acid sequencethat is at least 95% homologous to a fragment of a nucleotide sequencethat encodes SEQ ID NO:6; a nucleic acid sequence that is at least 95%homologous to a fragment of a nucleotide sequence that encodes SEQ IDNO:8; a nucleic acid sequence that is at least 95% homologous to afragment of a nucleotide sequence that encodes SEQ ID NO:18; a nucleicacid sequence that is at least 95% homologous to a fragment of anucleotide sequence that encodes SEQ ID NO:20; a nucleic acid sequencethat is at least 95% homologous to a fragment of a nucleotide sequencethat encodes SEQ ID NO:22; and a nucleic acid sequence that is at least95% homologous to a fragment of a nucleotide sequence that encodes SEQID NO:24.

In some embodiments the compositions include HPV E6-E7 fusion antigensselected from the group consisting of: nucleotide sequence that encodesSEQ ID NO:2; nucleotide sequence that encodes SEQ ID NO:4; nucleotidesequence that encodes SEQ ID NO:6; nucleotide sequence that encodes SEQID NO:8; nucleotide sequence that encodes SEQ ID NO:18; nucleotidesequence that encodes SEQ ID NO:20; nucleotide sequence that encodes SEQID NO:22; nucleotide sequence that encodes SEQ ID NO:24; a nucleic acidsequence that is at least 95% homologous to a nucleic acid sequencenucleotide sequence that encodes SEQ ID NO:2; a nucleic acid sequencethat is at least 95% homologous to a nucleotide sequence that encodesSEQ ID NO:4; a nucleic acid sequence that is at least 95% homologous toa nucleotide sequence that encodes SEQ ID NO:6; a nucleic acid sequencethat is at least 95% homologous to a nucleotide sequence that encodesSEQ ID NO:8; a nucleic acid sequence that is at least 95% homologous toa nucleotide sequence that encodes SEQ ID NO:18; a nucleic acid sequencethat is at least 95% homologous to a nucleotide sequence that encodesSEQ ID NO:20; a nucleic acid sequence that is at least 95% homologous toa nucleotide sequence that encodes SEQ ID NO:22; a nucleic acid sequencethat is at least 95% homologous to a nucleotide sequence that encodesSEQ ID NO:24; a fragment of a nucleotide sequence that encodes SEQ IDNO:2; a fragment of a nucleotide sequence that encodes SEQ ID NO:4; afragment of a nucleotide sequence that encodes SEQ ID NO:6; a fragmentof a nucleotide sequence that encodes SEQ ID NO:8; a fragment of anucleotide sequence that encodes SEQ ID NO:18; a fragment of anucleotide sequence that encodes SEQ ID NO:20; a fragment of anucleotide sequence that encodes SEQ ID NO:22; a fragment of anucleotide sequence that encodes SEQ ID NO:24; a nucleic acid sequencethat is at least 95% homologous to a fragment of a nucleotide sequencethat encodes SEQ ID NO:2; a nucleic acid sequence that is at least 95%homologous to a fragment of a nucleotide sequence that encodes SEQ IDNO:4; a nucleic acid sequence that is at least 95% homologous to afragment of a nucleotide sequence that encodes SEQ ID NO:6; a nucleicacid sequence that is at least 95% homologous to a fragment of anucleotide sequence that encodes SEQ ID NO:8; a nucleic acid sequencethat is at least 95% homologous to a fragment of a nucleotide sequencethat encodes SEQ ID NO:18; a nucleic acid sequence that is at least 95%homologous to a fragment of a nucleotide sequence that encodes SEQ IDNO:20; a nucleic acid sequence that is at least 95% homologous to afragment of a nucleotide sequence that encodes SEQ ID NO:22; and anucleic acid sequence that is at least 95% homologous to a fragment of anucleotide sequence that encodes SEQ ID NO:24.

In some embodiments the compositions include HPV E6-E7 fusion antigensselected from the group consisting of: nucleotide sequence that encodesSEQ ID NO:6; nucleotide sequence that encodes SEQ ID NO:8; nucleotidesequence that encodes SEQ ID NO:18; nucleotide sequence that encodes SEQID NO:20; nucleotide sequence that encodes SEQ ID NO:22; nucleotidesequence that encodes SEQ ID NO:24; a nucleic acid sequence that is atleast 95% homologous to a nucleotide sequence that encodes SEQ ID NO:6;a nucleic acid sequence that is at least 95% homologous to a nucleotidesequence that encodes SEQ ID NO:8; a nucleic acid sequence that is atleast 95% homologous to a nucleotide sequence that encodes SEQ ID NO:18;a nucleic acid sequence that is at least 95% homologous to a nucleotidesequence that encodes SEQ ID NO:20; a nucleic acid sequence that is atleast 95% homologous to a nucleotide sequence that encodes SEQ ID NO:22;a nucleic acid sequence that is at least 95% homologous to a nucleotidesequence that encodes SEQ ID NO:24; a fragment of a nucleotide sequencethat encodes SEQ ID NO:6; a fragment of a nucleotide sequence thatencodes SEQ ID NO:8; a fragment of a nucleotide sequence that encodesSEQ ID NO:18; a fragment of a nucleotide sequence that encodes SEQ IDNO:20; a fragment of a nucleotide sequence that encodes SEQ ID NO:22; afragment of a nucleotide sequence that encodes SEQ ID NO:24; a nucleicacid sequence that is at least 95% homologous to a fragment of anucleotide sequence that encodes SEQ ID NO:6; a nucleic acid sequencethat is at least 95% homologous to a fragment of a nucleotide sequencethat encodes SEQ ID NO:8; a nucleic acid sequence that is at least 95%homologous to a fragment of a nucleotide sequence that encodes SEQ IDNO:18; a nucleic acid sequence that is at least 95% homologous to afragment of a nucleotide sequence that encodes SEQ ID NO:20; a nucleicacid sequence that is at least 95% homologous to a fragment of anucleotide sequence that encodes SEQ ID NO:22; and a nucleic acidsequence that is at least 95% homologous to a fragment of a nucleotidesequence that encodes SEQ ID NO:24.

In some embodiments the compositions include HPV E6-E7 fusion antigensselected from the group consisting of: nucleotide sequence that encodesSEQ ID NO:2; nucleotide sequence that encodes SEQ ID NO:4; nucleotidesequence that encodes SEQ ID NO:22; nucleotide sequence that encodes SEQID NO:24; a nucleic acid sequence that is at least 95% homologous to anucleic acid sequence nucleotide sequence that encodes SEQ ID NO:2; anucleic acid sequence that is at least 95% homologous to a nucleotidesequence that encodes SEQ ID NO:4; a nucleic acid sequence that is atleast 95% homologous to a nucleotide sequence that encodes SEQ ID NO:22;a nucleic acid sequence that is at least 95% homologous to a nucleotidesequence that encodes SEQ ID NO:24; a fragment of a nucleotide sequencethat encodes SEQ ID NO:2; a fragment of a nucleotide sequence thatencodes SEQ ID NO:4; a fragment of a nucleotide sequence that encodesSEQ ID NO:22; a fragment of a nucleotide sequence that encodes SEQ IDNO:24; a nucleic acid sequence that is at least 95% homologous to afragment of a nucleotide sequence that encodes SEQ ID NO:2; a nucleicacid sequence that is at least 95% homologous to a fragment of anucleotide sequence that encodes SEQ ID NO:4; a nucleic acid sequencethat is at least 95% homologous to a fragment of a nucleotide sequencethat encodes SEQ ID NO:22; and a nucleic acid sequence that is at least95% homologous to a fragment of a nucleotide sequence that encodes SEQID NO:24.

In some embodiments the compositions include HPV E6-E7 fusion antigensselected from the group consisting of: nucleotide sequence that encodesSEQ ID NO:18; nucleotide sequence that encodes SEQ ID NO:20; nucleotidesequence that encodes SEQ ID NO:22; a nucleic acid sequence that is atleast 95% homologous to a nucleotide sequence that encodes SEQ ID NO:18;a nucleic acid sequence that is at least 95% homologous to a nucleotidesequence that encodes SEQ ID NO:20; a nucleic acid sequence that is atleast 95% homologous to a nucleotide sequence that encodes SEQ ID NO:22;a fragment of a nucleotide sequence that encodes SEQ ID NO:18; afragment of a nucleotide sequence that encodes SEQ ID NO:20; a fragmentof a nucleotide sequence that encodes SEQ ID NO:22; a nucleic acidsequence that is at least 95% homologous to a fragment of a nucleotidesequence that encodes SEQ ID NO:18; a nucleic acid sequence that is atleast 95% homologous to a fragment of a nucleotide sequence that encodesSEQ ID NO:20; and a nucleic acid sequence that is at least 95%homologous to a fragment of a nucleotide sequence that encodes SEQ IDNO:22.

In another aspect of the invention, there are provided compositionscomprising one or more nucleotide sequences encoding an HPV E6-E7 fusionantigen selected from the group consisting of: SEQ ID NO:1; SEQ ID NO:3;SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQID NO:23; a nucleic acid sequence that is at least 95% homologous to SEQID NO:1; a nucleic acid sequence that is at least 95% homologous to SEQID NO:3; a nucleic acid sequence that is at least 95% homologous to SEQID NO:5; a nucleic acid sequence that is at least 95% homologous to SEQID NO:7; a nucleic acid sequence that is at least 95% homologous to SEQID NO:17; a nucleic acid sequence that is at least 95% homologous to SEQID NO:19; a nucleic acid sequence that is at least 95% homologous to SEQID NO:21; a nucleic acid sequence that is at least 95% homologous to SEQID NO:23; a fragment of SEQ ID NO:1; a fragment of SEQ ID NO:3; afragment of SEQ ID NO:5; a fragment of SEQ ID NO:7; a fragment of SEQ IDNO:17; a fragment of SEQ ID NO:19; a fragment of SEQ ID NO:21; afragment of SEQ ID NO:23; a nucleic acid sequence that is at least 95%homologous to a fragment of SEQ ID NO:1; a nucleic acid sequence that isat least 95% homologous to a fragment of SEQ ID NO:3; a nucleic acidsequence that is at least 95% homologous to a fragment of SEQ ID NO:5; anucleic acid sequence that is at least 95% homologous to a fragment ofSEQ ID NO:7; a nucleic acid sequence that is at least 95% homologous toa fragment of SEQ ID NO:17; a nucleic acid sequence that is at least 95%homologous to a fragment of SEQ ID NO:19; a nucleic acid sequence thatis at least 95% homologous to a fragment of SEQ ID NO:21; and a nucleicacid sequence that is at least 95% homologous to a fragment of SEQ IDNO:23.

In some embodiments the compositions include HPV E6-E7 fusion antigensselected from the group consisting of: SEQ ID NO:5; SEQ ID NO:7; SEQ IDNO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; a nucleic acid sequencethat is at least 95% homologous to SEQ ID NO:5; a nucleic acid sequencethat is at least 95% homologous to SEQ ID NO:7; a nucleic acid sequencethat is at least 95% homologous to SEQ ID NO:17; a nucleic acid sequencethat is at least 95% homologous to SEQ ID NO:19; a nucleic acid sequencethat is at least 95% homologous to SEQ ID NO:21; a nucleic acid sequencethat is at least 95% homologous to SEQ ID NO:23; a fragment of SEQ IDNO:5; a fragment of SEQ ID NO:7; a fragment of SEQ ID NO:17; a fragmentof SEQ ID NO:19; a fragment of SEQ ID NO:21; a fragment of SEQ ID NO:23;a nucleic acid sequence that is at least 95% homologous to a fragment ofSEQ ID NO:5; a nucleic acid sequence that is at least 95% homologous toa fragment of SEQ ID NO:7; a nucleic acid sequence that is at least 95%homologous to a fragment of SEQ ID NO:17; a nucleic acid sequence thatis at least 95% homologous to a fragment of SEQ ID NO:19; a nucleic acidsequence that is at least 95% homologous to a fragment of SEQ ID NO:21;and a nucleic acid sequence that is at least 95% homologous to afragment of SEQ ID NO:23.

In some embodiments the compositions include HPV E6-E7 fusion antigensselected from the group consisting of: SEQ ID NO:1; SEQ ID NO:3; SEQ IDNO:21; SEQ ID NO:23; a nucleic acid sequence that is at least 95%homologous to SEQ ID NO:1; a nucleic acid sequence that is at least 95%homologous to SEQ ID NO:3; a nucleic acid sequence that is at least 95%homologous to SEQ ID NO:21; a nucleic acid sequence that is at least 95%homologous to SEQ ID NO:23; a fragment of SEQ ID NO:1; a fragment of SEQID NO:3; a fragment of SEQ ID NO:21; a fragment of SEQ ID NO:23; anucleic acid sequence that is at least 95% homologous to a fragment ofSEQ ID NO:1; a nucleic acid sequence that is at least 95% homologous toa fragment of SEQ ID NO:3; a nucleic acid sequence that is at least 95%homologous to a fragment of SEQ ID NO:21; and a nucleic acid sequencethat is at least 95% homologous to a fragment of SEQ ID NO:23.

In some embodiments the compositions include HPV E6-E7 fusion antigensselected from the group consisting of: SEQ ID NO:17; SEQ ID NO:19; SEQID NO:21; a nucleic acid sequence that is at least 95% homologous to SEQID NO:17; a nucleic acid sequence that is at least 95% homologous to SEQID NO:19; a nucleic acid sequence that is at least 95% homologous to SEQID NO:21; a fragment of SEQ ID NO:17; a fragment of SEQ ID NO:19; afragment of SEQ ID NO:21; nucleic acid sequence that is at least 95%homologous to a fragment of SEQ ID NO:17; a nucleic acid sequence thatis at least 95% homologous to a fragment of SEQ ID NO:19; and a nucleicacid sequence that is at least 95% homologous to a fragment of SEQ IDNO:21.

In some embodiments the nucleotide sequences described herein is absentthe leader sequence. The nucleotide sequences comprising HPV6, HPV11,HPV16, HPV18, HPV31, HPV33, HPV39, HPV45, HPV52, and HPV58 is absent aleader sequence. In particular, the HPV E6-E7 fusion antigens includingnucleotide sequence that encodes SEQ ID NO:2; nucleotide sequence thatencodes SEQ ID NO:4; nucleotide sequence that encodes SEQ ID NO:6;nucleotide sequence that encodes SEQ ID NO:8; nucleotide sequence thatencodes SEQ ID NO:18; nucleotide sequence that encodes SEQ ID NO:20;nucleotide sequence that encodes SEQ ID NO:22; and nucleotide sequencethat encodes SEQ ID NO:24 are absent a leader sequence at 5′ end, forexample nucleotide sequence encoding SEQ ID NO.10. In particular, theHPV E6-E7 fusion antigens including nucleotide sequence SEQ ID NO:1; SEQID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:17; SEQ ID NO:19; SEQ IDNO:21; SEQ ID NO:23 are absent a leader sequence at 5′ end, for examplenucleotide sequence SEQ ID NO.9.

In some embodiments the nucleotide sequences of the present inventioncan be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous with the providednucleotide sequences; preferably 95%, 96%, 97%, 98%, or 99%; or 98% or99%.

The nucleotide sequences provided can be included into one of a varietyof known vectors or delivery systems, including a plasmid, viral vector,lipid vector, nanoparticle; preferably a plasmid.

In additional aspects, provided are pharmaceutical compositionscomprising the disclosed nucleotide sequences; preferably with multipleanitgens.

In some aspects, there are methods of inducing an effective immuneresponse in an individual against more than one subtype of HPV,comprising administering to said individual a composition comprising oneor more of the nucelotides sequences provided; preferably, thecompositions have more than one antigen. The methods preferably includea step of introducing the provided nucleotide sequences into theindividual by electroporation.

SEQ ID NO:1 comprises a nucleotide sequence that encodes a consensusimmunogen of HPV 6 E6 and E7 proteins. SEQ ID NO:1 includes an IgEleader sequence SEQ ID NO:9 linked to the nucleotide sequence at the 5′end of SEQ ID NO:1. SEQ ID NO:2 comprises the amino acid sequence forthe consensus immunogen of HPV 6 E6 and E7 proteins. SEQ ID NO:2includes an IgE leader sequence SEQ ID NO:10 at the N-terminal end ofthe consensus immunogen sequence. The IgE leader sequence is SEQ IDNO:10 and can be encoded by SEQ ID NO:9.

In some embodiments, vaccines include SEQ ID NO:2, or a nucleic acidmolecule that encodes SEQ ID NO:2.

Fragments of SEQ ID NO:2 may be 100% identical to the full length exceptmissing at least one amino acid from the N and/or C terminal, in eachcase with or without signal peptides and/or a methionine at position 1.Fragments of SEQ ID NO:2 can comprise 60% or more, 65% or more, 70% ormore, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more,92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% ormore, 98% or more, 99% or more percent of the length of the full lengthSEQ ID NO:2, excluding any heterologous signal peptide added. Thefragment can, preferably, comprise a fragment of SEQ ID NO:2 that is 95%or more, 96% or more, 97% or more, 98% or more or 99% or more homologousto SEQ ID NO:2 and additionally comprise an N terminal methionine orheterologous signal peptide which is not included when calculatingpercent homology Fragments can further comprise an N terminal methionineand/or a signal peptide such as an immunoglobulin signal peptide, forexample an IgE or IgG signal peptide. The N terminal methionine and/orsignal peptide may be linked to the fragment.

Fragments of a nucleic acid sequence SEQ ID NO:1 can be 100% identicalto the full length except missing at least one nucleotide from the 5′and/or 3′ end, in each case with or without sequences encoding signalpeptides and/or a methionine at position 1. Fragments can comprise 60%or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% ormore, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more,95% or more, 96% or more, 97% or more, 98% or more, 99% or more percentof the length of full length coding sequence SEQ ID NO:1, excluding anyheterologous signal peptide added. The fragment can, preferably,comprise a fragment that encodes a polypeptide that is 95% or more, 96%or more, 97% or more, 98% or more or 99% or more homologous to theantigen SEQ ID NO:2 and additionally optionally comprise sequenceencoding an N terminal methionine or heterologous signal peptide whichis not included when calculating percent homology Fragments can furthercomprise coding sequences for an N terminal methionine and/or a signalpeptide such as an immunoglobulin signal peptide, for example an IgE orIgG signal peptide. The coding sequence encoding the N terminalmethionine and/or signal peptide may be linked to the fragment.

In some embodiments, fragments of SEQ ID NO:1 may comprise 786 or morenucleotides; in some embodiments, 830 or more nucleotides; in someembodiments 856 or more nucleotides; and in some embodiments, 865 ormore nucleotides. In some embodiments, fragments of SEQ ID NO:1 such asthose set forth herein may further comprise coding sequences for the IgEleader sequences. In some embodiments, fragments of SEQ ID NO:1 do notcomprise coding sequences for the IgE leader sequences.

In some embodiments, fragments of SEQ ID NO:2 may comprise 252 or moreamino acids; in some embodiments, 266 or more amino acids; in someembodiments, 275 or more amino acids; and in some embodiments, 278 ormore amino acids.

SEQ ID NO:3 comprises a nucleotide sequence that encodes a consensusimmunogen of HPV 11 E6 and E7 proteins. SEQ ID NO:3 includes an IgEleader sequence SEQ ID NO:9 linked to the nucleotide sequence at the 5′end of SEQ ID NO:3. SEQ ID NO:4 comprises the amino acid sequence forthe consensus immunogen of HPV 11 E6 and E7 proteins. SEQ ID NO:4includes an IgE leader sequence SEQ ID NO:10 at the N-terminal end ofthe consensus immunogen sequence. The IgE leader sequence is SEQ IDNO:10 and can be encoded by SEQ ID NO:9.

In some embodiments, vaccines include SEQ ID NO:4, or a nucleic acidmolecule that encodes SEQ ID NO:4.

Fragments of SEQ ID NO:4 may be 100% identical to the full length exceptmissing at least one amino acid from the N and/or C terminal, in eachcase with or without signal peptides and/or a methionine at position 1.Fragments of SEQ ID NO:4 can comprise 60% or more, 65% or more, 70% ormore, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more,92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% ormore, 98% or more, 99% or more percent of the length of the full lengthSEQ ID NO:4, excluding any heterologous signal peptide added. Thefragment can, preferably, comprise a fragment of SEQ ID NO:4 that is 95%or more, 96% or more, 97% or more, 98% or more or 99% or more homologousto SEQ ID NO:4 and additionally comprise an N terminal methionine orheterologous signal peptide which is not included when calculatingpercent homology Fragments can further comprise an N terminal methionineand/or a signal peptide such as an immunoglobulin signal peptide, forexample an IgE or IgG signal peptide. The N terminal methionine and/orsignal peptide may be linked to the fragment.

Fragments of a nucleic acid sequence SEQ ID NO:3 can be 100% identicalto the full length except missing at least one nucleotide from the 5′and/or 3′ end, in each case with or without sequences encoding signalpeptides and/or a methionine at position 1. Fragments can comprise 60%or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% ormore, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more,95% or more, 96% or more, 97% or more, 98% or more, 99% or more percentof the length of full length coding sequence SEQ ID NO:3, excluding anyheterologous signal peptide added. The fragment can, preferably,comprise a fragment that encodes a polypeptide that is 95% or more, 96%or more, 97% or more, 98% or more or 99% or more homologous to theantigen SEQ ID NO:4 and additionally optionally comprise sequenceencoding an N terminal methionine or heterologous signal peptide whichis not included when calculating percent homology Fragments can furthercomprise coding sequences for an N terminal methionine and/or a signalpeptide such as an immunoglobulin signal peptide, for example an IgE orIgG signal peptide. The coding sequence encoding the N terminalmethionine and/or signal peptide may be linked to the fragment.

In some embodiments, fragments of SEQ ID NO:3 may comprise 786 or morenucleotides; in some embodiments, 830 or more nucleotides; in someembodiments 856 or more nucleotides; and in some embodiments, 865 ormore nucleotides. In some embodiments, fragments of SEQ ID NO:3 such asthose set forth herein may further comprise coding sequences for the IgEleader sequences. In some embodiments, fragments of SEQ ID NO:3 do notcomprise coding sequences for the IgE leader sequences.

In some embodiments, fragments of SEQ ID NO:4 may comprise 252 or moreamino acids; in some embodiments, 266 or more amino acids; in someembodiments, 275 or more amino acids; and in some embodiments, 278 ormore amino acids.

SEQ ID NO:5 comprises a nucleotide sequence that encodes a consensusimmunogen of HPV 33 E6 and E7 proteins. SEQ ID NO:5 includes an IgEleader sequence SEQ ID NO:9 linked to the nucleotide sequence at the 5′end of SEQ ID NO:5. SEQ ID NO:6 comprises the amino acid sequence forthe consensus immunogen of HPV 33 E6 and E7 proteins. SEQ ID NO:6includes an IgE leader sequence SEQ ID NO:10 at the N-terminal end ofthe consensus immunogen sequence. The IgE leader sequence is SEQ IDNO:10 and can be encoded by SEQ ID NO:9.

In some embodiments, vaccines include SEQ ID NO:6, or a nucleic acidmolecule that encodes SEQ ID NO:6.

Fragments of SEQ ID NO:6 may be 100% identical to the full length exceptmissing at least one amino acid from the N and/or C terminal, in eachcase with or without signal peptides and/or a methionine at position 1.Fragments of SEQ ID NO:6 can comprise 60% or more, 65% or more, 70% ormore, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more,92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% ormore, 98% or more, 99% or more percent of the length of the full lengthSEQ ID NO:6, excluding any heterologous signal peptide added. Thefragment can, preferably, comprise a fragment of SEQ ID NO:6 that is 95%or more, 96% or more, 97% or more, 98% or more or 99% or more homologousto SEQ ID NO:6 and additionally comprise an N terminal methionine orheterologous signal peptide which is not included when calculatingpercent homology Fragments can further comprise an N terminal methionineand/or a signal peptide such as an immunoglobulin signal peptide, forexample an IgE or IgG signal peptide. The N terminal methionine and/orsignal peptide may be linked to the fragment.

Fragments of a nucleic acid sequence SEQ ID NO:5 can be 100% identicalto the full length except missing at least one nucleotide from the 5′and/or 3′ end, in each case with or without sequences encoding signalpeptides and/or a methionine at position 1. Fragments can comprise 60%or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% ormore, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more,95% or more, 96% or more, 97% or more, 98% or more, 99% or more percentof the length of full length coding sequence SEQ ID NO:5, excluding anyheterologous signal peptide added. The fragment can, preferably,comprise a fragment that encodes a polypeptide that is 95% or more, 96%or more, 97% or more, 98% or more or 99% or more homologous to theantigen SEQ ID NO:6 and additionally optionally comprise sequenceencoding an N terminal methionine or heterologous signal peptide whichis not included when calculating percent homology Fragments can furthercomprise coding sequences for an N terminal methionine and/or a signalpeptide such as an immunoglobulin signal peptide, for example an IgE orIgG signal peptide. The coding sequence encoding the N terminalmethionine and/or signal peptide may be linked to the fragment.

In some embodiments, fragments of SEQ ID NO:5 may comprise 746 or morenucleotides; in some embodiments, 787 or more nucleotides; in someembodiments 812 or more nucleotides; and in some embodiments, 820 ormore nucleotides. In some embodiments, fragments of SEQ ID NO:5 such asthose set forth herein may further comprise coding sequences for the IgEleader sequences. In some embodiments, fragments of SEQ ID NO:5 do notcomprise coding sequences for the IgE leader sequences.

In some embodiments, fragments of SEQ ID NO:6 may comprise 235 or moreamino acids; in some embodiments, 248 or more amino acids; in someembodiments, 256 or more amino acids; and in some embodiments, 259 ormore amino acids.

SEQ ID NO:7 comprises a nucleotide sequence that encodes a consensusimmunogen of HPV 58 E6 and E7 proteins. SEQ ID NO:7 includes an IgEleader sequence SEQ ID NO:9 linked to the nucleotide sequence at the 5′end of SEQ ID NO:7. SEQ ID NO:8 comprises the amino acid sequence forthe consensus immunogen of HPV 58 E6 and E7 proteins. SEQ ID NO:8includes an IgE leader sequence SEQ ID NO:10 at the N-terminal end ofthe consensus immunogen sequence. The IgE leader sequence is SEQ IDNO:10 and can be encoded by SEQ ID NO:9.

In some embodiments, vaccines include SEQ ID NO:8, or a nucleic acidmolecule that encodes SEQ ID NO:8.

Fragments of SEQ ID NO:8 may be 100% identical to the full length exceptmissing at least one amino acid from the N and/or C terminal, in eachcase with or without signal peptides and/or a methionine at position 1.Fragments of SEQ ID NO:8 can comprise 60% or more, 65% or more, 70% ormore, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more,92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% ormore, 98% or more, 99% or more percent of the length of the full lengthSEQ ID NO:8, excluding any heterologous signal peptide added. Thefragment can, preferably, comprise a fragment of SEQ ID NO:8 that is 95%or more, 96% or more, 97% or more, 98% or more or 99% or more homologousto SEQ ID NO:8 and additionally comprise an N terminal methionine orheterologous signal peptide which is not included when calculatingpercent homology Fragments can further comprise an N terminal methionineand/or a signal peptide such as an immunoglobulin signal peptide, forexample an IgE or IgG signal peptide. The N terminal methionine and/orsignal peptide may be linked to the fragment.

Fragments of a nucleic acid sequence SEQ ID NO:7 can be 100% identicalto the full length except missing at least one nucleotide from the 5′and/or 3′ end, in each case with or without sequences encoding signalpeptides and/or a methionine at position 1. Fragments can comprise 60%or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% ormore, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more,95% or more, 96% or more, 97% or more, 98% or more, 99% or more percentof the length of full length coding sequence SEQ ID NO:7, excluding anyheterologous signal peptide added. The fragment can, preferably,comprise a fragment that encodes a polypeptide that is 95% or more, 96%or more, 97% or more, 98% or more or 99% or more homologous to theantigen SEQ ID NO:8 and additionally optionally comprise sequenceencoding an N terminal methionine or heterologous signal peptide whichis not included when calculating percent homology Fragments can furthercomprise coding sequences for an N terminal methionine and/or a signalpeptide such as an immunoglobulin signal peptide, for example an IgE orIgG signal peptide. The coding sequence encoding the N terminalmethionine and/or signal peptide may be linked to the fragment.

In some embodiments, fragments of SEQ ID NO:7 may comprise 752 or morenucleotides; in some embodiments, 794 or more nucleotides; in someembodiments 819 or more nucleotides; and in some embodiments, 827 ormore nucleotides. In some embodiments, fragments of SEQ ID NO:7 such asthose set forth herein may further comprise coding sequences for the IgEleader sequences. In some embodiments, fragments of SEQ ID NO:7 do notcomprise coding sequences for the IgE leader sequences.

In some embodiments, fragments of SEQ ID NO:8 may comprise 235 or moreamino acids; in some embodiments, 249 or more amino acids; in someembodiments, 257 or more amino acids; and in some embodiments, 260 ormore amino acids.

SEQ ID NO:17 comprises a nucleotide sequence that encodes a consensusimmunogen of HPV 31 E6 and E7 proteins. SEQ ID NO:17 includes an IgEleader sequence SEQ ID NO:9 linked to the nucleotide sequence at the 5′end of SEQ ID NO:17. SEQ ID NO:18 comprises the amino acid sequence forthe consensus immunogen of HPV 31 E6 and E7 proteins. SEQ ID NO:18includes an IgE leader sequence SEQ ID NO:10 at the N-terminal end ofthe consensus immunogen sequence. The IgE leader sequence is SEQ IDNO:10 and can be encoded by SEQ ID NO:9.

In some embodiments, vaccines include SEQ ID NO:18, or a nucleic acidmolecule that encodes SEQ ID NO:18.

Fragments of SEQ ID NO:18 may be 100% identical to the full lengthexcept missing at least one amino acid from the N and/or C terminal, ineach case with or without signal peptides and/or a methionine atposition 1. Fragments of SEQ ID NO:18 can comprise 60% or more, 65% ormore, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more,91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% ormore, 97% or more, 98% or more, 99% or more percent of the length of thefull length SEQ ID NO:18, excluding any heterologous signal peptideadded. The fragment can, preferably, comprise a fragment of SEQ ID NO:18that is 95% or more, 96% or more, 97% or more, 98% or more or 99% ormore homologous to SEQ ID NO:18 and additionally comprise an N terminalmethionine or heterologous signal peptide which is not included whencalculating percent homology Fragments can further comprise an Nterminal methionine and/or a signal peptide such as an immunoglobulinsignal peptide, for example an IgE or IgG signal peptide. The N terminalmethionine and/or signal peptide may be linked to the fragment.

Fragments of a nucleic acid sequence SEQ ID NO:17 can be 100% identicalto the full length except missing at least one nucleotide from the 5′and/or 3′ end, in each case with or without sequences encoding signalpeptides and/or a methionine at position 1. Fragments can comprise 60%or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% ormore, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more,95% or more, 96% or more, 97% or more, 98% or more, 99% or more percentof the length of full length coding sequence SEQ ID NO:17, excluding anyheterologous signal peptide added. The fragment can, preferably,comprise a fragment that encodes a polypeptide that is 95% or more, 96%or more, 97% or more, 98% or more or 99% or more homologous to theantigen SEQ ID NO:18 and additionally optionally comprise sequenceencoding an N terminal methionine or heterologous signal peptide whichis not included when calculating percent homology Fragments can furthercomprise coding sequences for an N terminal methionine and/or a signalpeptide such as an immunoglobulin signal peptide, for example an IgE orIgG signal peptide. The coding sequence encoding the N terminalmethionine and/or signal peptide may be linked to the fragment.

In some embodiments, fragments of SEQ ID NO:17 may comprise 713 or morenucleotides; in some embodiments, 752 or more nucleotides; in someembodiments 776 or more nucleotides; and in some embodiments, 784 ormore nucleotides. In some embodiments, fragments of SEQ ID NO:17 such asthose set forth herein may further comprise coding sequences for the IgEleader sequences. In some embodiments, fragments of SEQ ID NO:17 do notcomprise coding sequences for the IgE leader sequences.

In some embodiments, fragments of SEQ ID NO:18 may comprise 236 or moreamino acids; in some embodiments, 249 or more amino acids; in someembodiments, 257 or more amino acids; and in some embodiments, 259 ormore amino acids.

SEQ ID NO:19 comprises a nucleotide sequence that encodes a consensusimmunogen of HPV 52 E6 and E7 proteins. SEQ ID NO:19 includes an IgEleader sequence SEQ ID NO:9 linked to the nucleotide sequence at the 5′end of SEQ ID NO:19. SEQ ID NO:20 comprises the amino acid sequence forthe consensus immunogen of HPV 52 E6 and E7 proteins. SEQ ID NO:20includes an IgE leader sequence SEQ ID NO:10 at the N-terminal end ofthe consensus immunogen sequence. The IgE leader sequence is SEQ IDNO:10 and can be encoded by SEQ ID NO:9.

In some embodiments, vaccines include SEQ ID NO:20, or a nucleic acidmolecule that encodes SEQ ID NO:20.

Fragments of SEQ ID NO:20 may be 100% identical to the full lengthexcept missing at least one amino acid from the N and/or C terminal, ineach case with or without signal peptides and/or a methionine atposition 1. Fragments of SEQ ID NO:20 can comprise 60% or more, 65% ormore, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more,91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% ormore, 97% or more, 98% or more, 99% or more percent of the length of thefull length SEQ ID NO:20, excluding any heterologous signal peptideadded. The fragment can, preferably, comprise a fragment of SEQ ID NO:20that is 95% or more, 96% or more, 97% or more, 98% or more or 99% ormore homologous to SEQ ID NO:20 and additionally comprise an N terminalmethionine or heterologous signal peptide which is not included whencalculating percent homology Fragments can further comprise an Nterminal methionine and/or a signal peptide such as an immunoglobulinsignal peptide, for example an IgE or IgG signal peptide. The N terminalmethionine and/or signal peptide may be linked to the fragment.

Fragments of a nucleic acid sequence SEQ ID NO:19 can be 100% identicalto the full length except missing at least one nucleotide from the 5′and/or 3′ end, in each case with or without sequences encoding signalpeptides and/or a methionine at position 1. Fragments can comprise 60%or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% ormore, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more,95% or more, 96% or more, 97% or more, 98% or more, 99% or more percentof the length of full length coding sequence SEQ ID NO:19, excluding anyheterologous signal peptide added. The fragment can, preferably,comprise a fragment that encodes a polypeptide that is 95% or more, 96%or more, 97% or more, 98% or more or 99% or more homologous to theantigen SEQ ID NO:20 and additionally optionally comprise sequenceencoding an N terminal methionine or heterologous signal peptide whichis not included when calculating percent homology Fragments can furthercomprise coding sequences for an N terminal methionine and/or a signalpeptide such as an immunoglobulin signal peptide, for example an IgE orIgG signal peptide. The coding sequence encoding the N terminalmethionine and/or signal peptide may be linked to the fragment.

In some embodiments, fragments of SEQ ID NO:19 may comprise 713 or morenucleotides; in some embodiments, 752 or more nucleotides; in someembodiments 776 or more nucleotides; and in some embodiments, 784 ormore nucleotides. In some embodiments, fragments of SEQ ID NO:19 such asthose set forth herein may further comprise coding sequences for the IgEleader sequences. In some embodiments, fragments of SEQ ID NO:19 do notcomprise coding sequences for the IgE leader sequences.

In some embodiments, fragments of SEQ ID NO:20 may comprise 236 or moreamino acids; in some embodiments, 249 or more amino acids; in someembodiments, 257 or more amino acids; and in some embodiments, 259 ormore amino acids.

SEQ ID NO:21 comprises a nucleotide sequence that encodes a consensusimmunogen of HPV16 E6 and E7 proteins. SEQ ID NO:21 includes an IgEleader sequence SEQ ID NO:9 linked to the nucleotide sequence at the 5′end of SEQ ID NO:21. SEQ ID NO:22 comprises the amino acid sequence forthe consensus immunogen of HPV16 E6 and E7 proteins. SEQ ID NO:22includes an IgE leader sequence SEQ ID NO:10 at the N-terminal end ofthe consensus immunogen sequence. The IgE leader sequence is SEQ IDNO:10 and can be encoded by SEQ ID NO:9.

In some embodiments, vaccines include SEQ ID NO:22, or a nucleic acidmolecule that encodes SEQ ID NO:22.

Fragments of SEQ ID NO:22 may be 100% identical to the full lengthexcept missing at least one amino acid from the N and/or C terminal, ineach case with or without signal peptides and/or a methionine atposition 1. Fragments of SEQ ID NO:22 can comprise 60% or more, 65% ormore, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more,91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% ormore, 97% or more, 98% or more, 99% or more percent of the length of thefull length SEQ ID NO:22, excluding any heterologous signal peptideadded. The fragment can, preferably, comprise a fragment of SEQ ID NO:22that is 95% or more, 96% or more, 97% or more, 98% or more or 99% ormore homologous to SEQ ID NO:22 and additionally comprise an N terminalmethionine or heterologous signal peptide which is not included whencalculating percent homology Fragments can further comprise an Nterminal methionine and/or a signal peptide such as an immunoglobulinsignal peptide, for example an IgE or IgG signal peptide. The N terminalmethionine and/or signal peptide may be linked to the fragment.

Fragments of a nucleic acid sequence SEQ ID NO:21 can be 100% identicalto the full length except missing at least one nucleotide from the 5′and/or 3′ end, in each case with or without sequences encoding signalpeptides and/or a methionine at position 1. Fragments can comprise 60%or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% ormore, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more,95% or more, 96% or more, 97% or more, 98% or more, 99% or more percentof the length of full length coding sequence SEQ ID NO:21, excluding anyheterologous signal peptide added. The fragment can, preferably,comprise a fragment that encodes a polypeptide that is 95% or more, 96%or more, 97% or more, 98% or more or 99% or more homologous to theantigen SEQ ID NO:22 and additionally optionally comprise sequenceencoding an N terminal methionine or heterologous signal peptide whichis not included when calculating percent homology Fragments can furthercomprise coding sequences for an N terminal methionine and/or a signalpeptide such as an immunoglobulin signal peptide, for example an IgE orIgG signal peptide. The coding sequence encoding the N terminalmethionine and/or signal peptide may be linked to the fragment.

In some embodiments, fragments of SEQ ID NO:21 may comprise 736 or morenucleotides; in some embodiments, 777 or more nucleotides; in someembodiments 802 or more nucleotides; and in some embodiments, 810 ormore nucleotides. In some embodiments, fragments of SEQ ID NO:21 such asthose set forth herein may further comprise coding sequences for the IgEleader sequences. In some embodiments, fragments of SEQ ID NO:21 do notcomprise coding sequences for the IgE leader sequences.

In some embodiments, fragments of SEQ ID NO:22 may comprise 238 or moreamino acids; in some embodiments, 251 or more amino acids; in someembodiments, 259 or more amino acids; and in some embodiments, 261 ormore amino acids.

SEQ ID NO:23 comprises a nucleotide sequence that encodes a consensusimmunogen of HPV18 E6 and E7 proteins. SEQ ID NO:23 includes an IgEleader sequence SEQ ID NO:9 linked to the nucleotide sequence at the 5′end of SEQ ID NO:23. SEQ ID NO:24 comprises the amino acid sequence forthe consensus immunogen of HPV18 E6 and E7 proteins. SEQ ID NO:24includes an IgE leader sequence SEQ ID NO:10 at the N-terminal end ofthe consensus immunogen sequence. The IgE leader sequence is SEQ IDNO:10 and can be encoded by SEQ ID NO:9.

In some embodiments, vaccines include SEQ ID NO:24, or a nucleic acidmolecule that encodes SEQ ID NO:24.

Fragments of SEQ ID NO:24 may be 100% identical to the full lengthexcept missing at least one amino acid from the N and/or C terminal, ineach case with or without signal peptides and/or a methionine atposition 1. Fragments of SEQ ID NO:24 can comprise 60% or more, 65% ormore, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more,91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% ormore, 97% or more, 98% or more, 99% or more percent of the length of thefull length SEQ ID NO:24, excluding any heterologous signal peptideadded. The fragment can, preferably, comprise a fragment of SEQ ID NO:24that is 95% or more, 96% or more, 97% or more, 98% or more or 99% ormore homologous to SEQ ID NO:24 and additionally comprise an N terminalmethionine or heterologous signal peptide which is not included whencalculating percent homology Fragments can further comprise an Nterminal methionine and/or a signal peptide such as an immunoglobulinsignal peptide, for example an IgE or IgG signal peptide. The N terminalmethionine and/or signal peptide may be linked to the fragment.

Fragments of a nucleic acid sequence SEQ ID NO:23 can be 100% identicalto the full length except missing at least one nucleotide from the 5′and/or 3′ end, in each case with or without sequences encoding signalpeptides and/or a methionine at position 1. Fragments can comprise 60%or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% ormore, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more,95% or more, 96% or more, 97% or more, 98% or more, 99% or more percentof the length of full length coding sequence SEQ ID NO:23, excluding anyheterologous signal peptide added. The fragment can, preferably,comprise a fragment that encodes a polypeptide that is 95% or more, 96%or more, 97% or more, 98% or more or 99% or more homologous to theantigen SEQ ID NO:24 and additionally optionally comprise sequenceencoding an N terminal methionine or heterologous signal peptide whichis not included when calculating percent homology Fragments can furthercomprise coding sequences for an N terminal methionine and/or a signalpeptide such as an immunoglobulin signal peptide, for example an IgE orIgG signal peptide. The coding sequence encoding the N terminalmethionine and/or signal peptide may be linked to the fragment

In some embodiments, fragments of SEQ ID NO:23 may comprise 705 or morenucleotides; in some embodiments, 744 or more nucleotides; in someembodiments 767 or more nucleotides; and in some embodiments, 775 ormore nucleotides. In some embodiments, fragments of SEQ ID NO:23 such asthose set forth herein may further comprise coding sequences for the IgEleader sequences. In some embodiments, fragments of SEQ ID NO:23 do notcomprise coding sequences for the IgE leader sequences.

In some embodiments, fragments of SEQ ID NO:24 may comprise 234 or moreamino acids; in some embodiments, 247 or more amino acids; in someembodiments, 255 or more amino acids; and in some embodiments, 257 ormore amino acids.

There are compositions comprising an amino acid sequence that isselected from SEQ ID NO:2; SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8; SEQ IDNO:18; SEQ ID NO:20; SEQ ID NO:22; or SEQ ID NO:24; or fragments thereofhaving at least 95% homology; or combinations thereof. In someembodiments, the compositions comprise SEQ ID NO:2; SEQ ID NO:4; SEQ IDNO:6, SEQ ID NO:8; SEQ ID NO:18; SEQ ID NO:20; SEQ ID NO:22; and SEQ IDNO:24. In some embodiments, the compositions comprise SEQ ID NO:6, SEQID NO:8; SEQ ID NO:18; SEQ ID NO:20; SEQ ID NO:22; and SEQ ID NO:24. Insome embodiments, the compositions comprise SEQ ID NO:2; SEQ ID NO:4;SEQ ID NO:22; and SEQ ID NO:24. In some embodiments, the compositionscomprise SEQ ID NO:18; SEQ ID NO:20; and SEQ ID NO:22.

The amino acid sequence can be fragments that have at least 95% homologywith any one of the amino acid sequence. In some embodiments, the aminoacid sequence can be fragments that have at least 98% homology with anyone of the amino acid sequence. In some embodiments, the amino acidsequence can be fragments that have at least 99% homology with any oneof the amino acid sequence.

According to some embodiments, methods of inducing an immune response inindividuals against an immunogen comprise administering to theindividual the amino acid sequence for a consensus immunogen selectedfrom the group consisting of HPV 6 E6 and E7, HPV 11 E6 and E7, HPV 16E6 and E7, HPV 18 E6 and E7, HPV 31 E6 and E7, HPV 33 E6 and E7, HPV 52E6 and E7, HPV 58 E6 and E7, HPV 45 E6 and E7, and HPV 39 E6 and E7,functional fragments thereof, and expressible coding sequences thereof.Preferably, the immunogens are consensus HPV6, HPV11, HPV16, HPV18,HPV31, HPV33, HPV52, and HPV58. Preferably, the immunogens are consensusHPV16, HPV18, HPV31, HPV33, HPV52, and HPV58. Preferably, the immunogensare consensus HPV6, HPV11, HPV16, and HPV18. Preferably, the immunogensare consensus HPV16, HPV31, and HPV52.

Some embodiments comprise an isolated nucleic acid molecule that encodesthe amino acid sequence for a consensus immunogen selected from thegroup consisting of HPV 6 E6 and E7, HPV 11 E6 and E7, HPV 16 E6 and E7,HPV 18 E6 and E7, HPV 31 E6 and E7, HPV 33 E6 and E7, HPV 52 E6 and E7,HPV 58 E6 and E7, HPV 45 E6 and E7, and HPV 39 E6 and E7, and fragmentsthereof. Some embodiments comprise a recombinant vaccine that encodesthe amino acid sequence for a consensus immunogen selected from thegroup consisting of HPV 6 E6 and E7, HPV 11 E6 and E7, HPV 16 E6 and E7,HPV 18 E6 and E7, HPV 31 E6 and E7, HPV 33 E6 and E7, HPV 52 E6 and E7,HPV 58 E6 and E7, HPV 45 E6 and E7, and HPV 39 E6 and E7, and fragmentsthereof. Some embodiments comprise a subunit vaccine that comprises theamino acid sequence for a consensus immunogen selected from the groupconsisting of HPV 6 E6 and E7, HPV 11 E6 and E7, HPV 16 E6 and E7, HPV18 E6 and E7, HPV 31 E6 and E7, HPV 33 E6 and E7, HPV 52 E6 and E7, HPV58 E6 and E7, HPV 45 E6 and E7, and HPV 39 E6 and E7, and fragmentsthereof. Some embodiments comprise a live attenuated vaccine and/or akilled vaccine that comprise the amino acid sequence for a consensusimmunogen selected from the group consisting of HPV 6 E6 and E7, HPV 11E6 and E7, HPV 16 E6 and E7, HPV 18 E6 and E7, HPV 31 E6 and E7, HPV 33E6 and E7, HPV 52 E6 and E7, HPV 58 E6 and E7, HPV 45 E6 and E7, and HPV39 E6 and E7.

There are methods of inducing an immune response in an individualagainst HPV comprising administering to said individual a compositioncomprising a nucleic acid sequences provided herein. In someembodiments, the methods also include introducing the nucleic acidsequences into the individual by electroporation.

In some aspects, there are methods of inducing an immune response in anindividual against HPV comprising administering to said individual acomposition comprising a amino acid sequence provided herein. In someembodiments, the methods also include introducing the amino acidsequences into the individual by electroporation.

In one aspect, the vaccines herein are those that elicit an immuneresponse against HPV subtypes found predominantly to be associated withforms of head and neck cancer, and other forms of otolaryngologicdiseases, in particular the vaccines include HPV 6 E6 and E7 and HPV 11E6 and E7, and preferably both.

In another aspect, the vaccines herein are those the elicit an immuneresponse against HPV subtypes found predominantly to be associated withforms of cervical cancer in patients ex-United States and moreparticularly patients in Asia, in particular the vaccines include HPV 33E6 and E7 and HPV 58 E6 and E7, and preferably both.

There are preferred combinations useful to elicit an immune responseagainst HPV subtypes found to be associated with cervical cancer,including precancerous lesions, which include: HPV subtypes 16, 18, 31,33, 52, 58, 6, 11, 39, and 45 or HPV subtypes 16, 18, 31, 33, 52, 58, 6,and 11. Other subcombinations for this cervical cancer include:

16 and 18; 16, 18, and 6; 16, 18, and 11; 16, 18, and 31; 16, 18, and33; 16, 18, and 52; 16, 18, and 58; 16, 18, 6, and 11; 16, 18, 6, and31; 16, 18, 6, and 33; 16, 18, 6, and 52, 16, 18, 6, and 58; 16, 18, 11and 31; 16, 18, 11 and 33, 16, 18, 11 and 52; 16, 18, 11 and 58; 16, 18,31 and 33; 16, 18, 31 and 52; 16, 18, 31 and 58; 16, 18, 33 and 52; 16,18, 33 and 58; 16, 18, 52 and 58;

6, 11, and 16; 6, 11, and 18; 6, 11, and 31; 6, 11, and 33; 6, 11, and52; 6, 11, and 58; 6, 11, 16, and 31; 6, 11, 16, and 33; 6, 11, 16, and52; 6, 11, 16, and 58; 6, 11, 18, and 31; 6, 11, 18, and 33; 6, 11, 18,and 52; 6, 11, 18, and 58; 6, 11, 31, and 33; 6, 11, 31, and 52; 6, 11,31, and 58; 6, 11, 33, and 52; 6, 11, 33, and 58; 6, 11, 52, and 58;

6, 16, and 31; 6, 16, and 33; 6, 16, and 52; 6, 16, and 58; 6, 16, 31and 33; 6, 16, 31 and 52; 6, 16, 31 and 58; 6, 16, 33 and 52; 6, 16, 33and 58; 6, 16, 52 and 58;

6, 18 and 31; 6, 18 and 33; 6, 18 and 52; 6, 18 and 58; 6, 18, 31 and33; 6, 18, 31 and 52; 6, 18, 31 and 58; 6, 18, 33 and 52; 6, 18, 33 and58;

6, 31 and 33; 6, 31 and 52; 6, 31 and 58; 6, 31, 33 and 52; 6, 31, 33and 58;

6, 33, and 52; 6, 33, and 58; 6, 33, 52 and 58;

6, 52 and 58;

11, 31 and 33; 11, 31 and 52; 11, 31 and 58; 11, 31, 33 and 52; 11, 31,33 and 58; 11, 31, 52 and 58;

11, 31 and 52; 11, 31 and 58; 11, 31, 52 and 58;

11, 52, and 58;

16, 31 and 33; 16, 31, and 52; 16, 31, and 58; 16, 31, 33, and 52; 16,31, 52, and 58;

16, 33, and 52; 16, 33, and 58; 16, 33, 52 and 58;

18, 31, and 33; 18, 31, and 52; 18, 31, and 58; 18, 31, 33 and 52; 18,31, 33 and 58;

31, 33 and 52; 31, 33 and 58; 31, 33, 52 and 58; and

31, 52, and 58.

Improved vaccines comprise proteins and genetic constructs that encodeproteins with epitopes that make them particularly effective asimmunogens against which anti-HPV immune responses can be induced.Accordingly, vaccines can be provided to induce a therapeutic orprophylactic immune response. In some embodiments, the means to deliverthe immunogen is a DNA vaccine, a recombinant vaccine, a protein subunitvaccine, a composition comprising the immunogen, an attenuated vaccineor a killed vaccine. In some embodiments, the vaccine comprises acombination selected from the groups consisting of: one or more DNAvaccines, one or more recombinant vaccines, one or more protein subunitvaccines, one or more compositions comprising the immunogen, one or moreattenuated vaccines and one or more killed vaccines.

Aspects of the invention provide methods of delivering the codingsequences of the protein on nucleic acid molecule such as plasmid, aspart of recombinant vaccines and as part of attenuated vaccines, asisolated proteins or proteins part of a vector.

According to some aspects of the present invention, compositions andmethods are provided which prophylactically and/or therapeuticallyimmunize an individual.

DNA vaccines are described in U.S. Pat. Nos. 5,593,972, 5,739,118,5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055,5,676,594, and the priority applications cited therein, which are eachincorporated herein by reference. In addition to the delivery protocolsdescribed in those applications, alternative methods of delivering DNAare described in U.S. Pat. Nos. 4,945,050 and 5,036,006, which are bothincorporated herein by reference.

The present invention relates to improved attenuated live vaccines,improved killed vaccines and improved vaccines that use recombinantvectors to deliver foreign genes that encode antigens and well assubunit and glycoprotein vaccines. Examples of attenuated live vaccines,those using recombinant vectors to deliver foreign antigens, subunitvaccines and glycoprotein vaccines are described in U.S. Pat. Nos.4,510,245; 4,797,368; 4,722,848; 4,790,987; 4,920,209; 5,017,487;5,077,044; 5,110,587; 5,112,749; 5,174,993; 5,223,424; 5,225,336;5,240,703; 5,242,829; 5,294,441; 5,294,548; 5,310,668; 5,387,744;5,389,368; 5,424,065; 5,451,499; 5,453,364; 5,462,734; 5,470,734;5,474,935; 5,482,713; 5,591,439; 5,643,579; 5,650,309; 5,698,202;5,955,088; 6,034,298; 6,042,836; 6,156,319 and 6,589,529, which are eachincorporated herein by reference.

When taken up by a cell, the genetic construct(s) may remain present inthe cell as a functioning extrachromosomal molecule and/or integrateinto the cell's chromosomal DNA. DNA may be introduced into cells whereit remains as separate genetic material in the form of a plasmid orplasmids. Alternatively, linear DNA that can integrate into thechromosome may be introduced into the cell. When introducing DNA intothe cell, reagents that promote DNA integration into chromosomes may beadded. DNA sequences that are useful to promote integration may also beincluded in the DNA molecule. Alternatively, RNA may be administered tothe cell. It is also contemplated to provide the genetic construct as alinear minichromosome including a centromere, telomeres and an origin ofreplication. Gene constructs may remain part of the genetic material inattenuated live microorganisms or recombinant microbial vectors whichlive in cells. Gene constructs may be part of genomes of recombinantviral vaccines where the genetic material either integrates into thechromosome of the cell or remains extrachromosomal. Genetic constructsinclude regulatory elements necessary for gene expression of a nucleicacid molecule. The elements include: a promoter, an initiation codon, astop codon, and a polyadenylation signal. In addition, enhancers areoften required for gene expression of the sequence that encodes thetarget protein or the immunomodulating protein. It is necessary thatthese elements be operable linked to the sequence that encodes thedesired proteins and that the regulatory elements are operably in theindividual to whom they are administered.

Initiation codons and stop codon are generally considered to be part ofa nucleotide sequence that encodes the desired protein. However, it isnecessary that these elements are functional in the individual to whomthe gene construct is administered. The initiation and terminationcodons must be in frame with the coding sequence.

Promoters and polyadenylation signals used must be functional within thecells of the individual.

Examples of promoters useful to practice the present invention,especially in the production of a genetic vaccine for humans, includebut are not limited to promoters from Simian Virus 40 (SV40), MouseMammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (MV)such as the BIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV,Cytomegalovirus (CMV) such as the CMV immediate early promoter, EpsteinBarr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters fromhuman genes such as human Actin, human Myosin, human Hemoglobin, humanmuscle creatine and human metalothionein.

Examples of polyadenylation signals useful to practice the presentinvention, especially in the production of a genetic vaccine for humans,include but are not limited to SV40 polyadenylation signals and LTRpolyadenylation signals. In particular, the SV40 polyadenylation signalthat is in pCEP4 plasmid (Invitrogen, San Diego Calif.), referred to asthe SV40 polyadenylation signal, is used.

In addition to the regulatory elements required for DNA expression,other elements may also be included in the DNA molecule. Such additionalelements include enhancers. The enhancer may be selected from the groupincluding but not limited to: human Actin, human Myosin, humanHemoglobin, human muscle creatine and viral enhancers such as those fromCMV, RSV and EBV.

Genetic constructs can be provided with mammalian origin of replicationin order to maintain the construct extrachromosomally and producemultiple copies of the construct in the cell. Plasmids pVAX1, pCEP4 andpREP4 from Invitrogen (San Diego, Calif.) contain the Epstein Barr virusorigin of replication and nuclear antigen EBNA-1 coding region whichproduces high copy episomal replication without integration.

In some preferred embodiments related to immunization applications,nucleic acid molecule(s) are delivered which include nucleotidesequences that encode protein of the invention, and, additionally, genesfor proteins which further enhance the immune response against suchtarget proteins. Examples of such genes are those which encode othercytokines and lymphokines such as alpha-interferon, gamma-interferon,platelet derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermalgrowth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18,MHC, CD80, CD86 and IL-15 including IL-15 having the signal sequencedeleted and optionally including the signal peptide from IgE. Othergenes which may be useful include those encoding: MCP-1, MIP-1α, MIP-1p,IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1,MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3,CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L,vascular growth factor, IL-7, nerve growth factor, vascular endothelialgrowth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP,Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, CaspaseICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB,Inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFkB, Bax,TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND,Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F,TAP1, TAP2 and functional fragments thereof

An additional element may be added which serves as a target for celldestruction if it is desirable to eliminate cells receiving the geneticconstruct for any reason. A herpes thymidine kinase (tk) gene in anexpressible form can be included in the genetic construct. The druggangcyclovir can be administered to the individual and that drug willcause the selective killing of any cell producing tk, thus, providingthe means for the selective destruction of cells with the geneticconstruct.

In order to maximize protein production, regulatory sequences may beselected which are well suited for gene expression in the cells theconstruct is administered into. Moreover, codons may be selected whichare most efficiently transcribed in the cell. One having ordinary skillin the art can produce DNA constructs that are functional in the cells.

In some embodiments, gene constructs may be provided in which the codingsequences for the proteins described herein are linked to IgE signalpeptide. In some embodiments, proteins described herein are linked toIgE signal peptide.

In some embodiments for which protein is used, for example, one havingordinary skill in the art can, using well known techniques, produce andisolate proteins of the invention using well known techniques. In someembodiments for which protein is used, for example, one having ordinaryskill in the art can, using well known techniques, inserts DNA moleculesthat encode a protein of the invention into a commercially availableexpression vector for use in well known expression systems. For example,the commercially available plasmid pSE420 (Invitrogen, San Diego,Calif.) may be used for production of protein in E. coli. Thecommercially available plasmid pYES2 (Invitrogen, San Diego, Calif.)may, for example, be used for production in S. cerevisiae strains ofyeast. The commercially available MAXBAC™ complete baculovirusexpression system (Invitrogen, San Diego, Calif.) may, for example, beused for production in insect cells. The commercially available plasmidpcDNA I or pcDNA3 (Invitrogen, San Diego, Calif.) may, for example, beused for production in mammalian cells such as Chinese Hamster Ovarycells. One having ordinary skill in the art can use these commercialexpression vectors and systems or others to produce protein by routinetechniques and readily available starting materials. (See e.g., Sambrooket al., Molecular Cloning a Laboratory Manual, Second Ed. Cold SpringHarbor Press (1989) which is incorporated herein by reference.) Thus,the desired proteins can be prepared in both prokaryotic and eukaryoticsystems, resulting in a spectrum of processed forms of the protein.

One having ordinary skill in the art may use other commerciallyavailable expression vectors and systems or produce vectors using wellknown methods and readily available starting materials. Expressionsystems containing the requisite control sequences, such as promotersand polyadenylation signals, and preferably enhancers are readilyavailable and known in the art for a variety of hosts. See e.g.,Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. ColdSpring Harbor Press (1989). Genetic constructs include the proteincoding sequence operably linked to a promoter that is functional in thecell line into which the constructs are transfected. Examples ofconstitutive promoters include promoters from cytomegalovirus or SV40.Examples of inducible promoters include mouse mammary leukemia virus ormetallothionein promoters. Those having ordinary skill in the art canreadily produce genetic constructs useful for transfecting with cellswith DNA that encodes protein of the invention from readily availablestarting materials. The expression vector including the DNA that encodesthe protein is used to transform the compatible host which is thencultured and maintained under conditions wherein expression of theforeign DNA takes place.

The protein produced is recovered from the culture, either by lysing thecells or from the culture medium as appropriate and known to those inthe art. One having ordinary skill in the art can, using well knowntechniques, isolate protein that is produced using such expressionsystems. The methods of purifying protein from natural sources usingantibodies which specifically bind to a specific protein as describedabove may be equally applied to purifying protein produced byrecombinant DNA methodology.

In addition to producing proteins by recombinant techniques, automatedpeptide synthesizers may also be employed to produce isolated,essentially pure protein. Such techniques are well known to those havingordinary skill in the art and are useful if derivatives which havesubstitutions not provided for in DNA-encoded protein production.

The nucleic acid molecules may be delivered using any of several wellknown technologies including DNA injection (also referred to as DNAvaccination), recombinant vectors such as recombinant adenovirus,recombinant adenovirus associated virus and recombinant vaccinia.

Routes of administration include, but are not limited to, intramuscular,intransally, intraperitoneal, intradermal, subcutaneous, intravenous,intraarterially, intraoccularly and oral as well as topically,transdermally, by inhalation or suppository or to mucosal tissue such asby lavage to vaginal, rectal, urethral, buccal and sublingual tissue.Preferred routes of administration include intramuscular,intraperitoneal, intradermal and subcutaneous injection. Geneticconstructs may be administered by means including, but not limited to,electroporation methods and devices, traditional syringes, needlelessinjection devices, or “microprojectile bombardment gone guns”.

Examples of electroporation devices and electroporation methodspreferred for facilitating delivery of the DNA vaccines, include thosedescribed in U.S. Pat. No. 7,245,963 by Draghia-Akli, et al., U.S.Patent Pub. 2005/0052630 submitted by Smith, et al., the contents ofwhich are hereby incorporated by reference in their entirety. Alsopreferred, are electroporation devices and electroporation methods forfacilitating delivery of the DNA vaccines provided in co-pending andco-owned U.S. patent application Ser. No. 11/874,072, filed Oct. 17,2007, which claims the benefit under 35 USC 119(e) to U.S. ProvisionalApplications Ser. No. 60/852,149, filed Oct. 17, 2006, and 60/978,982,filed Oct. 10, 2007, all of which are hereby incorporated in theirentirety.

The following is an example of an embodiment using electroporationtechnology, and is discussed in more detail in the patent referencesdiscussed above: electroporation devices can be configured to deliver toa desired tissue of a mammal a pulse of energy producing a constantcurrent similar to a preset current input by a user. The electroporationdevice comprises an electroporation component and an electrode assemblyor handle assembly. The electroporation component can include andincorporate one or more of the various elements of the electroporationdevices, including: controller, current waveform generator, impedancetester, waveform logger, input element, status reporting element,communication port, memory component, power source, and power switch.The electroporation component can function as one element of theelectroporation devices, and the other elements are separate elements(or components) in communication with the electroporation component. Insome embodiments, the electroporation component can function as morethan one element of the electroporation devices, which can be incommunication with still other elements of the electroporation devicesseparate from the electroporation component. The use of electroporationtechnology to deliver the improved HPV vaccine is not limited by theelements of the electroporation devices existing as parts of oneelectromechanical or mechanical device, as the elements can function asone device or as separate elements in communication with one another.The electroporation component is capable of delivering the pulse ofenergy that produces the constant current in the desired tissue, andincludes a feedback mechanism. The electrode assembly includes anelectrode array having a plurality of electrodes in a spatialarrangement, wherein the electrode assembly receives the pulse of energyfrom the electroporation component and delivers same to the desiredtissue through the electrodes. At least one of the plurality ofelectrodes is neutral during delivery of the pulse of energy andmeasures impedance in the desired tissue and communicates the impedanceto the electroporation component. The feedback mechanism can receive themeasured impedance and can adjust the pulse of energy delivered by theelectroporation component to maintain the constant current.

In some embodiments, the plurality of electrodes can deliver the pulseof energy in a decentralized pattern. In some embodiments, the pluralityof electrodes can deliver the pulse of energy in the decentralizedpattern through the control of the electrodes under a programmedsequence, and the programmed sequence is input by a user to theelectroporation component. In some embodiments, the programmed sequencecomprises a plurality of pulses delivered in sequence, wherein eachpulse of the plurality of pulses is delivered by at least two activeelectrodes with one neutral electrode that measures impedance, andwherein a subsequent pulse of the plurality of pulses is delivered by adifferent one of at least two active electrodes with one neutralelectrode that measures impedance.

In some embodiments, the feedback mechanism is performed by eitherhardware or software. Preferably, the feedback mechanism is performed byan analog closed-loop circuit. Preferably, this feedback occurs every 50μs, 20 μs, 10 μs or 1 μs, but is preferably a real-time feedback orinstantaneous (i.e., substantially instantaneous as determined byavailable techniques for determining response time). In someembodiments, the neutral electrode measures the impedance in the desiredtissue and communicates the impedance to the feedback mechanism, and thefeedback mechanism responds to the impedance and adjusts the pulse ofenergy to maintain the constant current at a value similar to the presetcurrent. In some embodiments, the feedback mechanism maintains theconstant current continuously and instantaneously during the delivery ofthe pulse of energy.

In some embodiments, the nucleic acid molecule is delivered to the cellsin conjunction with administration of a polynucleotide function enhanceror a genetic vaccine facilitator agent. Polynucleotide functionenhancers are described in U.S. Pat. No. 5,593,972, 5,962,428 andInternational Application Serial Number PCT/US94/00899 filed Jan. 26,1994, which are each incorporated herein by reference. Genetic vaccinefacilitator agents are described in US. Serial Number 021,579 filed Apr.1, 1994, which is incorporated herein by reference. The co-agents thatare administered in conjunction with nucleic acid molecules may beadministered as a mixture with the nucleic acid molecule or administeredseparately simultaneously, before or after administration of nucleicacid molecules. In addition, other agents which may functiontransfecting agents and/or replicating agents and/or inflammatory agentsand which may be co-administered with a GVF include growth factors,cytokines and lymphokines such as a-interferon, gamma-interferon,GM-CSF, platelet derived growth factor (PDGF), TNF, epidermal growthfactor (EGF), IL-1, IL-2, IL-4, IL-6, IL-10, IL-12 and IL-15 as well asfibroblast growth factor, surface active agents such asimmune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPSanalog including monophosphoryl Lipid A (WL), muramyl peptides, quinoneanalogs and vesicles such as squalene and squalene, and hyaluronic acidmay also be used administered in conjunction with the genetic constructIn some embodiments, an immunomodulating protein may be used as a GVF.In some embodiments, the nucleic acid molecule is provided inassociation with PLG to enhance delivery/uptake.

The pharmaceutical compositions according to the present inventioncomprise about 1 nanogram to about 2000 micrograms of DNA. In somepreferred embodiments, pharmaceutical compositions according to thepresent invention comprise about 5 nanogram to about 1000 micrograms ofDNA. In some preferred embodiments, the pharmaceutical compositionscontain about 10 nanograms to about 800 micrograms of DNA. In somepreferred embodiments, the pharmaceutical compositions contain about 0.1to about 500 micrograms of DNA. In some preferred embodiments, thepharmaceutical compositions contain about 1 to about 350 micrograms ofDNA. In some preferred embodiments, the pharmaceutical compositionscontain about 25 to about 250 micrograms of DNA. In some preferredembodiments, the pharmaceutical compositions contain about 100 to about200 microgram DNA.

The pharmaceutical compositions according to the present invention areformulated according to the mode of administration to be used. In caseswhere pharmaceutical compositions are injectable pharmaceuticalcompositions, they are sterile, pyrogen free and particulate free. Anisotonic formulation is preferably used. Generally, additives forisotonicity can include sodium chloride, dextrose, mannitol, sorbitoland lactose. In some cases, isotonic solutions such as phosphatebuffered saline are preferred. Stabilizers include gelatin and albumin.In some embodiments, a vasoconstriction agent is added to theformulation.

According to some embodiments of the invention, methods of inducingimmune responses are provided. The vaccine may be a protein based, liveattenuated vaccine, a cell vaccine, a recombinant vaccine or a nucleicacid or DNA vaccine. In some embodiments, methods of inducing an immuneresponse in individuals against an immunogen, including methods ofinducing mucosal immune responses, comprise administering to theindividual one or more of CTACK protein, TECK protein, MEC protein andfunctional fragments thereof or expressible coding sequences thereof incombination with an isolated nucleic acid molecule that encodes proteinof the invention and/or a recombinant vaccine that encodes protein ofthe invention and/or a subunit vaccine that protein of the inventionand/or a live attenuated vaccine and/or a killed vaccine. The one ormore of CTACK protein, TECK protein, MEC protein and functionalfragments thereof may be administered prior to, simultaneously with orafter administration of the isolated nucleic acid molecule that encodesan immunogen; and/or recombinant vaccine that encodes an immunogenand/or subunit vaccine that comprises an immunogen and/or liveattenuated vaccine and/or killed vaccine. In some embodiments, anisolated nucleic acid molecule that encodes one or more proteins ofselected from the group consisting of: CTACK, TECK, MEC and functionalfragments thereof is administered to the individual.

The present invention is further illustrated in the following Example.It should be understood that this Example, while indicating embodimentsof the invention, is given by way of illustration only. From the abovediscussion and this Example, one skilled in the art can ascertain theessential characteristics of this invention, and without departing fromthe spirit and scope thereof, can make various changes and modificationsof the invention to adapt it to various usages and conditions. Thus,various modifications of the invention in addition to those shown anddescribed herein will be apparent to those skilled in the art from theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

Each of the U.S. Patents, U.S. Applications, and references citedthroughout this disclosure are hereby incorporated in their entirety byreference.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, various modifications of the invention in addition tothose shown and described herein will be apparent to those skilled inthe art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Example 1

Novel Engineered HPV-16 DNA Vaccine Encoding a E6/E7 Fusion Protein

The immunogen has been designed to be expressed as a polyprotein wherebyE6 and E7 sequences are separated by a proteolytic cleavage site. Thepolyprotein is also expressed with an IgE leader sequence. Thepolyprotein design includes deletions or mutations in the E6 sequencewhich are important for p53 binding and degradation and mutations inribosome binding site on the E7 protein.

Coding sequences of HPV16 E6/E7 (SEQ ID NO: 21) encoding the polyprotein(SEQ ID NO:22) were inserted into the vector pVAX to generate anexpression plasmid. The optimized human papilloma virus 16-6&7 antigens(HPV16 E6&7), driven by the CMV promoter (PCMV) with the bovine growthhonnone 3′ end and poly-adenylation signal (bGHpA) using a pVAX backbonethat includes-the kanamycin resistance gene (Kan) and plasmid origin ofreplication (pUC ori).

HPV18 E6/E7 Vaccine Design and Expression

An optimized consensus sequences for human papilloma virus (HPV) viralproteins 18 E6&7 was prepared. The sequence is designed for high levelsof expression. This sequence is useful in our genetic immunizationtechnology. Results from experiments performed using the consensussequence were positive. The plasmid construct includes the nucleic acidsequence of the consensus HPV 18-6&7 (SEQ ID NO: 23). The nucleic acidsequence of the consensus HPV 18-6&7 that is incorporated into theplasmid includes coding sequence for the IgE leader peptide linked tothe coding sequences of the consensus HPV 18-6&7.

A expression plasmid pGX3002 was generated that expresses the optimizedhuman papilloma virus 18-6&7 antigens (HPVI8 E6&7) (SEQ ID NO:24),driven by the CMV promoter (PCMV) with the bovine growth honnone 3′ endand poly-adenylation signal (bGHpA) using a pVAX backbone thatincludes-the kanamycin resistance gene (Kan) and plasmid origin ofreplication (pUC ori).

HPV6 and HPV11 E6/E7 Vaccine Design and Expression

Construction of HPV6 and 11 E6/E7 Consensus-Based Fusion Immunogens

The HPV type 6 or 11 E6 and E7 gene sequences were collected fromGeneBank, and the consensus E6 and E7 nucleotide sequences were obtainedafter performing multiple alignment. The consensus sequence of HPV 6 E6or E7 proteins was generated from 98 or 20 sequences, respectively,while the consensus sequence of HPV 11 E6 or E7 proteins was generatedfrom 76 or 13 sequences, respectively. The multiple alignment procedureapplied in the phylogenetic study included the application of Clustal X(version 2.0). As indicated in FIGS. 1A and B, there were about 0-2% ofsequence divergence among the HPV strains belonging to the same type intheir E6 and E7 proteins. However, the genetic distances could go up to19.3% in the E6 protein and 16.3% in the E7 protein between HPV 6 and11. Based on these results from the phylogenic analyses, we developedtwo type-specific E6/E7 consensus DNA vaccines.

Several modifications were conducted after generating the consensusE6/E7 fusion sequence (FIG. 1C). A highly efficient leader sequence wasfused in frame upstream of the start codon to facilitate the expression.The codon and RNA optimization was also performed as describedpreviously (J. Yan, et al., Cellular immunity induced by a novel HPV18DNA vaccine encoding an E6/E7 fusion consensus protein in mice andrhesus macaques, Vaccine. 26 (2008) 5210-5215; and J. Yan, et al.,Induction of antitumor immunity in vivo following delivery of a novelHPV-16 DNA vaccine encoding an E6/E7 fusion antigen, Vaccine. 27 (2009)431-440). An endoproteolytic cleavage site was introduced between E6 andE7 protein for proper protein folding and better CTL processing. Bothsynthetic engineered 6E6E7 gene and 11E6E7 genes were 840 bp in length.The sequence verified synthetic genes were subcloned into the pVAXexpression vector at the BamHI and XhoI sites respectively for furtherstudy. The consensus amino acid sequences were obtained by translatingthe consensus nucleotide sequences.

After obtaining HPV 6 and 11 consensus E6 and E7 sequences, condonoptimization and RNA optimization was performed as previously described(J. Yan, et al., Vaccine. 26 (2008) 5210-5215; and J. Yan, et al.,Induction, Vaccine. 27 (2009) 431-440). The fusion genes encoding eitherHPV type 6 or 11 consensus E6/E7 fusion protein (6E6E7 or 11E6E7) weresynthesized and sequence verified. The synthesized 6E6E7 or 11E6E7 wasdigested with BamHI and XhoI, cloned into the expression vector pVAX(Invitrogen) under the control of the cytomegalovirus immediate-earlypromoter and these constructs were named as p6E6E7 or p11E6E7.

293-T cells were cultured in 6-well plates and transfected with pVAX,p6E6E7, or p11E6E7 using FuGENE6 Transfection Reagent (Roche AppliedScience, Indianapolis, Ind.). Two days after transfection, the cellswere lysed using Modified RIPA Cell Lysis Buffer and cell lysate wascollected. The Western blot analyses were performed with an anti-HAmonoclonal antibody (Sigma-Aldrich, St. Louis, Mo.) and visualized withhorseradish peroxidase-conjugated goat anti-mouse IgG (Sigma-Aldrich, StLouis, Mo.) using an ECL™ Western blot analysis system (Amersham,Piscataway, N.J.).

Indirect immunofluorescent assays were performed using humanrhabdomyosarcoma cells (RD cells) to verify expression of p6E6E7 andp11E6E7. RD cells cultured in chamber slides were transfected with pVAX,p6E6E7, or p11E6E7 using Turbofectin 8.0 (Origene, Rockville, Md.).Afterwards, the cells were fixed with PFA and permeabilized with 0.1%Triton-X in PBS. The cells were subjected to 1-2 hour incubations withprimary and secondary antibodies in addition to washes of PBSsupplemented with Glycine and BSA in between incubations. The primaryand secondary antibodies used were monoclonal mouse anti-HA(Sigma-Aldrich, St. Louis, Mo.) and FITC-conjugated anti-mouse IgG(Abcam, Cambridge, Mass.), respectively. Hoechst staining was alsoperformed to identify cell nuclei and localize the RD cells. After allincubations were completed, the cells were mounted with a glass slidefixed with Fluoromount-G (Southern Biotech, Birmingham, Ala.). Thesamples were viewed and imaged using a confocal microscope (CDBMicroscopy Core, University of Pennsylvania Cell and DevelopmentalBiology, Philadelphia, Pa.).

HPV 31, 33, 39, 45, 52 and 58 E6E7 Antigens

The HPV type 31, 33, 39, 45, 52, and 58 E6 and E7 gene sequences,individually, were collected from GeneBank, and the consensus E6 and E7nucleotide sequences were obtained after performing multiple alignment(type31—SEQ ID NO: 18; type33—SEQ ID NO:6; type52—SEQ ID NO:20; andtype58—SEQ ID NO:8). The multiple alignment procedure applied in thephylogenetic study included the application of Clustal X (version 2.0).Based on the results from the phylogenic analyses, we developedtype-specific E6/E7 consensus DNA vaccines.

Several modifications were conducted after generating the consensusE6/E7 fusion sequence. A highly efficient leader sequence was fused inframe upstream of the start codon to facilitate the expression. Thecodon and RNA optimization was also performed as described previously(J. Yan, et al., Cellular immunity induced by a novel HPV18 DNA vaccineencoding an E6/E7 fusion consensus protein in mice and rhesus macaques,Vaccine. 26 (2008) 5210-5215; and J. Yan, et al., Induction of antitumorimmunity in vivo following delivery of a novel HPV-16 DNA vaccineencoding an E6/E7 fusion antigen, Vaccine. 27 (2009) 431-440). Anendoproteolytic cleavage site was introduced between E6 and E7 proteinfor proper protein folding and better CTL processing. The sequenceverified synthetic genes were subcloned into the pVAX expression vectorat the BamHI and XhoI sites respectively for further study. Theconsensus amino acid sequences were obtained by translating theconsensus nucleotide sequences.

An expression plasmid was generated that expresses the optimized humanpapilloma virus 31, 33, 52, and 58-E6&7 antigens (HPV31 E6&7—SEQ IDNO:17, HPV33 E6&7—SEQ ID NO:5, HPV52 E6&7—SEQ ID NO:19, and HPV58E6&7—SEQ ID NO:7), driven by the CMV promoter (PCMV) with the bovinegrowth honnone 3′ end and poly-adenylation signal (bGHpA) using a pVAXbackbone that includes-the kanamycin resistance gene (Kan) and plasmidorigin of replication (pUC ori). Likewise, an expression plasmid isgenerated including an insert that is the optimized human papillomavirus 39 and 45 E6&E7 antigens.

Example 2

Mice and Treatment Groups Vaccinations

Female C57BL/6 mice between 6 to 8 weeks old were used in thisexperiment. Mice were obtained from the Jackson Laboratory (Bar Harbor,Me.). The mice were housed and maintained by the University LaboratoryAnimal Resources at the University of Pennsylvania (Philadelphia, Pa.)in observance with the policies of the National Institutes of Health andthe University of Pennsylvania Institutional Animal Care and UseCommittee (IACUC). The mice used in these experiments were separatedinto groups of four for immunization. Mice were immunized with p6E6E7,p11E6E7, or both constructs and pVAX group served as negative control.

DNA Vaccination and Electroporation

Each mouse received three doses of 20 μg of each DNA plasmid at 14-dayintervals. Mice in the group receiving both p6E6E7 and p11E6E7 received20 μg of both plasmids for a total of 40 μg of DNA per vaccination. TheDNA constructs were administered via intramuscular injection of theright quadriceps muscle, followed by square-wave pulses generated by theCELLECTRA™ electroporator (Inovio Pharmaceuticals, Blue Bell, Pa.). Theelectroporator was configured to deliver two electric pulses at 0.2 Ampsat 52 ms/pulse spaced apart by a 1 second delay. Electroporationprocedure was performed as described previously [12,13].

IFN-γ ELISpot Assay

Mice in both treatment and control groups were sacrificed 1 week afterthe third immunization. Spleens were harvested from each mouse andtransferred to RPMI-1640 medium with 10% FBS and antibiotics (R10).Using a stomacher (Seward Laboratory Systems, Bohemia, N.Y.), thespleens were pulverized and subsequently transferred through a cellstrainer and suspended in ACK lysing buffer. After removingerythrocytes, the splenocytes were isolated and suspended in R10 medium.High-protein IP 96-well Multiscreen™ plates (Millipore, Bedford, Mass.)were pre-coated with monoclonal mouse IFN-γ Capture Antibody (R&DSystems, Minneapolis, Minn.) and incubated overnight at 4° C. Afterthree washes with 1×PBS, the plates were blocked with 1% BSA and 5%sucrose in 1×PBS for 2 hours at ambient temperature. Isolatedsplenocytes in R10 medium were counted and added in triplicate wells at2×105 cells per well. Two sets of peptides spanning the consensus E6/E7sequence for HPV6 and HPV11 were reconstituted in DMSO (GenScript USA,Piscataway, N.J.). The peptides contained 15 amino acid sequences, ofwhich 8 residues overlapped with each sequential peptide. The peptidesfor HPV6 and 11 were each divided into two pools—one pool for E6 andanother for E7—at concentrations of 2 μg/mL in DMSO. Wells reserved forpositive and negative control received Concavalin A (Sigma-Aldrich, St.Louis, Mo.) and R10 culture medium in lieu of peptides, respectively.Plates were subsequently placed in a 5% CO2 atmosphere incubator. Afterincubation for 24 hours at 37° C., the wells were washed with 1×PBS.Biotinylated anti-mouse IFN-γ Detection Antibody (R&D Systems,Minneapolis, Minn.) was added to each well and then incubated overnightat 4° C. The plates were subsequently washed and processed per a colordevelopment protocol provided by R&D Systems using Streptavidin-AP andBCIP/NBT Plus (R&D Systems, Minneapolis, Minn.). The wells wereair-dried overnight and spots inside wells were scanned and counted byan ELISpot plate reader system with ImmunoSpot®3 and ImmunoSpot®4software (Cellular Technology Ltd., Shaker Heights, Ohio). Reportedspot-forming cell counts were converted to represent spot-forming unitsper 1×106 splenocytes using arithmetic.

Given their sensitivity and ability to illustrate T-cell activity, IFN-γELISpot assays were used to determine the number of antigen-specificIFN-gamma secreting cells in response to stimulation with either HPV 6or 11 E6 and E7 peptides.

As shown in FIGS. 3A and 3B, the average number of SFU/106 splenocytesfor mice vaccinated with p6E6E7 was 1442.8, while the average number ofSFU/106 for mice immunized with p11E6E7 was 2845, which were allsignificantly greater than the negative control group. Therefore, bothp6E6E7 and p11E6E7 were effective in eliciting robust type-specific E6and E7-specific immune response in mice.

Interestingly, the cross-reactive cellular immune responses were alsoinduced by vaccination with p6E6E7 or p11E6E7. The additive frequency ofSFU/106 splenocytes in p11E6E7 immunized mice against HPV 6 E6/E7peptides was 552.8 SFU/106 splenocytes, and the HPV 11 E6/E7-specificimmune responses in p6E6E7 immunized mice was 888.3 SFU/106 splenocytes.The E6 proteins of HPV 6 or 11 share about 80% identity, while the E7proteins of HPV6 or 11 shared about 84% identity. There may be someshared immune epitopes between HPV 6 or 11 E6 and E7 antigens. Thecross-reactivity observed from the IFN-gamma ELISpot assay indicatedthat there were shared immune epitopes between HPV6 and 11 E6 and E7antigens.

Splenocytes from mice that received both p6E6E7 and p11E6E7 (combogroup) were also subjected to the above ELISpot assays in order toexamine whether there was any immune interference when these twoconstructs were vaccinated together (FIGS. 3A and B). The combo groupexhibited an average of 1670 SFU/106 splenocytes (σ=55.7, p<0.001)against HPV6 E6 and E7 peptides. The same group of splenocytes produced2010 SFU/106 splenocytes (σ=247.8, p=0.002) against HPV11 E6 and E7peptides. The data suggest that concurrent vaccination with the twoconstructs can elicit a statistically significant E6 and E7-specificcellular response against HPV6 and HPV11 and that these responses do notinterfere with each other.

Epitope Mapping

Epitope mapping studies were performed to determine dominant epitopeswithin peptide pools, in order to determine the immune dominant peptideswithin the E6/E7 consensus antigens (FIGS. 4A and 4B). The studies wereperformed similarly to the previously mentioned IFN-γ ELISpot assay.Instead of pools, individual peptides were used to stimulate thesplenocytes.

Each peptide used in this single-peptide analysis represented apartially overlapping fragment of the E6 and E7 antigens of HPV6 orHPV11. The mapping data indicated that peptide 7 (TAEIYSYAYKQLKVL) SEQID NO:11 was the dominant epitope for the HPV6 E6 and E7 immunogens(FIG. 4A). TAEIYSYAYKQLKVL SEQ ID NO:11 contained 8, 9, 10-mer aminoacid epitopes that are verified to be an H2-Kb restricted by theHLA-binding prediction software made available by NIH BIMAS. To furtherdescribe the HPV11 E6 and E7-specific T-cell immune response (FIG. 4B).Peptide analysis was also performed with overlapping fragments of HPV11E6 and E7. Epitope mapping showed that the dominant epitopes for HPV 11E6 and E7 antigens were peptides 7 (TAEIYAYAYKNLKVV) SEQ ID NO:12 and 27(HCYEQLEDSSEDEVD) SEQ ID NO:13. As with peptide 7 in the HPV6 epitopemapping assay, the BIMAS HLA-binding prediction software confirmedTAEIYAYAYKNLKVV SEQ ID NO:12 to be an H2-Kb restricted epitope. AnotherHLA-binding peptide database, the Immune Epitope Database and AnalysisResource provided by NIH NIAID, confirmed that HCYEQLEDSSEDEVD SEQ IDNO:13 is an H2-Kb restricted epitope. Three immune subdominant peptides.numbers 6 (FCKNALTTAEIYSYA) SEQ ID NO:14, 9 (LFRGGYPYAACACCL) SEQ IDNO:15, and 13 (YAGYATTVEEETKQD) SEQ ID NO:16, were identified throughthis epitope mapping study.

Intracellular Cytokine Staining

In light of the high immune response portrayed by the IFN-γ ELISpotassays, intracellular cytokine staining assays were performed to providea more holistic overview of the cellular response induced by p6E6E7 andp11E6E7. Splenocytes from vaccinated and naive mouse groups wereisolated and stimulated with peptides spanning the E6 and E7 regions ofHPV6 and HPV11 for 4 hours at 37° C. in a 5% CO2 environment. Positiveand negative controls were used in the assay by placing cells in phorbol12-myristate 13-acetate (PMA) and R10 cell media, respectively. Afterincubation, the cells were first stained with ViViD Dye (Invitrogen,Carlsbad, Calif.) to differentiate between live and dead cells, then allcells were stained with the following surface marker antibodies: APC-Cy7Hamster anti-Mouse CD3e, PerCP-Cy5.5 Rat anti-Mouse CD4, and APC Ratanti-Mouse CD8a (BD Biosciences, San Diego, Calif.). The cells weresubsequently fixed using the Cytofix/Cytoperm kit (BD Biosciences, SanDiego, Calif.). After fixation per manufacturer protocol, the cells werestained with the following intracellular marker antibodies: Alexa Fluor700 Rat anti-Mouse IFN-γ, PE-Cy7 Rat anti-Mouse TNF Clone, and PE Ratanti-Mouse IL-2 (BD Biosciences, San Diego, Calif.). After staining, thecells were fixed with a solution of PBS containing 2% paraformaldehyde.The prepared cells were acquired using an LSR II flow cytometer equippedwith BD FACSDiva software (BD Biosciences, San Jose, Calif.). Acquireddata was analyzed using the latest version of FlowJo software (TreeStar, Ashland, Oreg.). CD4+ and CD8+ events were isolated using thefollowing sequence of gates: singlet from FSC-A vs FSC-H, allsplenocytes from FSC-A vs SSC-A, live cells from ViViD Dye (PacificBlue) vs SSC-A, CD3+ cells from CD3 (APC-Cy7) vs SSC-A, and CD4+ or CD8+from CD4 (PerCP-Cy5.5: positive—CD4+, negative—CD8+) vs SSC-A. The lasttwo populations were gated against Alexa Fluor 700, PE-Cy7, and PE toobserve changes in IL-2, IFN-γ, and TNF-α production, respectively.

Cells were gated such that intracellular cytokine staining data candifferentiate by CD4+ and CD8+ cell responses. When stimulated with HPV6E6 and E7-specific peptides, mice vaccinated with p6E6E7 exhibitedaverages of 0.163% (σ=0.09), 0.003% (σ=0.07), and 0.188% (σ=0.20) oftotal CD4+ cells producing IFN-γ, TNF-α, and IL-2, respectively (FIG.5A). The same group of mice had averages of 3.323% (σ=1.39), 0.838%(σ=0.32), and 1.172% (σ=1.81) of total CD8+ cells producing IFN-γ,TNF-α, and IL-2, respectively. The same intracellular cytokine data wascollected using splenocytes from mice vaccinated with p11E6E7 afterincubation with HPV11 E6 and E7 antigens (FIG. 5B). Of all CD4+ cells inthe p11E6E7 vaccinated mice, an average of 0.051% (σ=0.04) producedIFN-γ, 0.068% (σ=0.09) produced TNF-α, and 0.026% (σ=0.037) producedIL-2. Further, an average of 4.52% (σ=2.53), 2.08% (σ=1.56), and 0.21%(σ=0.22) of all CD8+ cells in p11E6E7 vaccinated mice produced IFN-γ,TNF-α, and IL-2, respectively. With the exception of a few panels, thepercentage of cytokine producing cells in treatment versus theirrespective naïve groups was statistically significant at a confidencelevel of 89% or higher. Observation of the magnitude of cytokineproduction of CD4+ cells versus CD8+ T-cells, one can conclude that theimmune responses elicited by p6E6E7 and p11E6E7 is heavily skewedtowards driving CD8+ lymphocytes, which are associated in all modelswith their cell clearance.

Statistical Analysis

Student's t tests were performed to analyze statistical significance ofall quantitative data produced in this study. Unless otherwiseindicated, p-values were calculated to determine statisticalsignificance at various confidence levels.

This study has shown compelling evidence that DNA vaccines may be ableto attain a level of immunogenicity found in experiments using otherpopular and traditional vaccine platforms. High levels of cellularresponses measured in other similar E6 and E7-specific HPV DNA vaccinestudies were associated with data suggestive of prophylactic andtherapeutic anti-tumor efficacy. Most HPV-related research and diseasechallenge models focus on cervical cancer. Given that HPV6 and HPV11 arenot as relevant to cervical cancer as other HPV serotypes, thetherapeutic efficacy of p6E6E7 and p11E6E7 cannot be fully evaluatedusing conventional HPV disease challenge models. It would be insightfulto determine the protective and therapeutic potential of p6E6E7 andp11E6E7 when appropriate disease challenge models become available.Thus, one can only infer the immunogenic efficacy of p6E6E7 and p11E6E7by looking at the high levels of IFN-γ and cytokine productionquantified by the ELISpot assays and intracellular cytokine staining.Nonetheless, such levels were found to be significantly robust inmagnitude and show promise that the two constructs would elicitsubstantive levels of cellular immune responses. Because HPV-relatedmalignancies are generally secondary to concurrent infection of multipleserotypes, there is a great need to look further into the feasibility ofcombining vaccines targeting different serotypes of the virus. Futurestudy looking into T-cell dynamics and vaccine competition is warrantedto further characterize the effects of concurrent vaccination of the twoplasmids.

Intracellular cytokine staining showed that vaccination with p6E6E7 andp11E6E7 was able to elicit a significant percentage of IFN-γ, TNF-α, andIL-2 producing T-cells. Given its currently known function in the immunesystem, IFN-γ has historically been used as a metric of cellular immuneresponses. Some of these important roles include the ability to modulateand stimulate innate and adaptive immunity. Moreover, it is widelyaccepted that the principle producers of IFN-γ are T-cells, making IFN-γproduction an acceptable mode of measuring the cellular immunogenicityof a given vaccine post-exposure to antigens. TNF-α is another cytokinethat is involved in the regulation of the immune system. Its knownability to induce apoptosis and regulate tumor proliferation makes it animportant parameter to consider when characterizing post-vaccinationimmune responses. TNF-α production may be of further interest given thepotential tumor proliferative properties of HPV6 and HPV11. IL-2 isanother signaling molecule that has been observed to play a central rolein the proliferation and differentiation of T-cells in the immunesystem. As a consequence, IL-2 is often examined in conjunction withother cytokines to gain further perspective on the magnitude and qualityof a particular immune response. Given the above, the significantpercentages of CD4+ and CD8+ cells producing IFN-γ, TNF-α, and IL-2after vaccination with p6E6E7 suggests that the vaccine was successfulin inducing a potent immune response. With the exception of IL-2secreting CD4+ cells, the same trend is true for cells isolated frommice vaccinated with p11E6E7. Moreover, it is noteworthy to observe thatCD8+ cells heavily drove the immune responses in vaccinated mice—acharacteristic that is significant in evaluating the anti-tumor efficacyof the two plasmids.

Example 3

Mice and Treatment Groups Vaccinations

Female C57BL/6 mice between 6 to 8 weeks old can be used. Mice can beobtained from the Jackson Laboratory (Bar Harbor, Me.). The mice can beseparated into groups of four for immunization. Mice can be immunizedwith one of the following combinations: a) plasmid encoding HPV16E6E7,plasmid encoding HPV18E6E7, plasmid encoding HPV6E6E7, plasmid encodingHPV11E6E7, plasmid encoding HPV31E6E7, plasmid encoding HPV33E6E7,plasmid encoding HPV52E6E7, and plasmid encoding HPV58E6E7; b) plasmidencoding HPV16E6E7, plasmid encoding HPV18E6E7, plasmid encoding plasmidencoding HPV31E6E7, plasmid encoding HPV33E6E7, plasmid encodingHPV52E6E7, and plasmid encoding HPV58E6E7; c) plasmid encodingHPV16E6E7, plasmid encoding HPV18E6E7, plasmid encoding HPV31E6E7, andplasmid encoding HPV33E6E7; d) plasmid encoding HPV16E6E7, plasmidencoding HPV18E6E7, plasmid encoding HPV6E6E7, and plasmid encodingHPV11E6E7; and pVAX group served as negative control.

DNA Vaccination and Electroporation

Each mouse received is given three doses of 20 μg of each DNA plasmidtotal at 14-day intervals. The DNA constructs are administered viaintramuscular injection of the right quadriceps muscle, followed bysquare-wave pulses generated by the CELLECTRA electroporator (InovioPharmaceuticals, Blue Bell, Pa.). The electroporator is configured todeliver two electric pulses at 0.2 Amps at 52 ms/pulse spaced apart by a1 second delay. Electroporation procedure was performed as describedpreviously, as in CELLECTRA information courtesy of InovioPharmaceuticals.

IFN-γ ELISpot Assay

Mice in both treatment and control groups are sacrificed 1 week afterthe third immunization. Spleens are harvested from each mouse andtransferred to RPMI-1640 medium with 10% FBS and antibiotics (R10).Using a stomacher (Seward Laboratory Systems, Bohemia, N.Y.), thespleens are pulverized and subsequently transferred through a cellstrainer and suspended in ACK lysing buffer. After removingerythrocytes, the splenocytes are isolated and suspended in R10 medium.High-protein IP 96-well Multiscreen plates (Millipore, Bedford, Mass.)are pre-coated with monoclonal mouse IFN-γ Capture Antibody (R&DSystems, Minneapolis, Minn.) and incubated overnight at 4° C. Afterthree washes with 1×PBS, the plates are blocked with 1% BSA and 5%sucrose in 1×PBS for 2 hours at ambient temperature. Isolatedsplenocytes in R10 medium are counted and added in triplicate wells at2×105 cells per well. Two sets of peptides spanning the consensus E6/E7sequence for each HPV plasmid are reconstituted in DMSO (GenScript USA,Piscataway, N.J.). The peptides contained 15 amino acid sequences, ofwhich 8 residues overlapped with each sequential peptide. The peptidesfor each HPV plasmid were each divided into two pools—one pool for E6and another for E7—at concentrations of 2 μg/mL in DMSO. Wells that arereserved for positive and negative control receive Concavalin A(Sigma-Aldrich, St. Louis, Mo.) and R10 culture medium in lieu ofpeptides, respectively. Plates are subsequently placed in a 5% CO2atmosphere incubator. After incubation for 24 hours at 37° C., the wellsare washed with 1—PBS. Biotinylated anti-mouse IFN-γ Detection Antibody(R&D Systems, Minneapolis, Minn.) is added to each well and thenincubated overnight at 4° C. The plates are subsequently washed andprocessed per a color development protocol provided by R&D Systems usingStreptavidin-AP and BCIP/NBT Plus (R&D Systems, Minneapolis, Minn.). Thewells are air-dried overnight and spots inside wells are scanned andcounted by an ELISpot plate reader system with ImmunoSpot®3 andImmunoSpot®4 software (Cellular Technology Ltd., Shaker Heights, Ohio).Reported spot-forming cell counts are converted to representspot-forming units per 1×106 splenocytes using arithmetic.

Given their sensitivity and ability to illustrate T-cell activity, IFN-γELISpot assays are used to determine the number of antigen-specificIFN-gamma secreting cells in response to stimulation with either HPV 6or 11 E6 and E7 peptides.

Example 4 Human Results from VGX-3100 Phase 1 (Combination of HPV16 E6E7and HPV18 E6E7)

Patients with Previously Treated CIN 2/3

IM delivery of VGX-3100 combination DNA vaccine using CELLECTRA®constant current EP device.

See Bagarazzi et al. Sci Transl Med 4, 155ra138 (2012), which isincorporated hereby in its entirety.

Number of Cohort Patient Dose (mg) 1 6 0.3 × 2 plasmids (HPV16/HPV18) 26 1 × 2 plasmids (HPV16/HPV18) 3 6 3 × 2 plasmids (HPV16/HPV18)Antibody Response:Antibodies against all 4 antigens with high titers in 15/18 (83%) andWestern Blot confirmation in all persist to 9 mos.Antigen-Specific Cellular Responses to HPV16,18 E6, E7:

-   -   14/18 (78%) POS by IFN-γ ELISpot (>50 SFU/106 PBMC)    -   Increase w/ dose up to >2500 SFU/106 PBMC for 1 Ag, >5,670        SFU/106 PBMC for all 4 antigens    -   5 subjects responded to all 4 antigens    -   Responses persist to 9 months after primary series    -   4th dose boosts T cell responses up to >2 years    -   HLA DR+/CD38+CD8+ T cells release Granzyme B/perforin for cell        killing

What is claimed is:
 1. A composition comprising at least one nucleotidesequence encoding at least one HPV E6-E7 fusion antigen from at leastone HPV subtype, wherein the at least one HPV subtype comprises HPV31;wherein the nucleotide sequence encoding the HPV31 E6-E7 fusion antigenencodes an amino acid sequence that is at least 98% homologous to SEQ IDNO:18.
 2. The composition of claim 1, further comprising at least onenucleotide sequence encoding at least one HPV E6-E7 fusion antigenselected from the group consisting of: nucleotide sequence that encodesSEQ ID NO:2; nucleotide sequence that encodes SEQ ID NO:4; nucleotidesequence that encodes SEQ ID NO:6; nucleotide sequence that encodes SEQID NO:8; nucleotide sequence that encodes SEQ ID NO:20; nucleotidesequence that encodes SEQ ID NO:22; nucleotide sequence that encodes SEQID NO:24; a nucleic acid sequence that is at least 95% homologous to anucleic acid sequence nucleotide sequence that encodes SEQ ID NO:2; anucleic acid sequence that is at least 95% homologous to a nucleotidesequence that encodes SEQ ID NO:4; a nucleic acid sequence that is atleast 95% homologous to a nucleotide sequence that encodes SEQ ID NO:6;a nucleic acid sequence that is at least 95% homologous to a nucleotidesequence that encodes SEQ ID NO:8; a nucleic acid sequence that is atleast 95% homologous to a nucleotide sequence that encodes SEQ ID NO:20;a nucleic acid sequence that is at least 95% homologous to a nucleotidesequence that encodes SEQ ID NO:22; and a nucleic acid sequence that isat least 95% homologous to a nucleotide sequence that encodes SEQ IDNO:24.
 3. The composition of claim 1, comprising one or more nucleotidesequences encoding an HPV E6-E7 fusion antigen selected from the groupconsisting of: SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQID NO: 17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; a nucleic acidsequence that is at least 95% homologous to SEQ ID NO:1; a nucleic acidsequence that is at least 95% homologous to SEQ ID NO:3; a nucleic acidsequence that is at least 95% homologous to SEQ ID NO:5; a nucleic acidsequence that is at least 95% homologous to SEQ ID NO:7; a nucleic acidsequence that is at least 95% homologous to SEQ ID NO: 17; a nucleicacid sequence that is at least 95% homologous to SEQ ID NO: 19; anucleic acid sequence that is at least 95% homologous to SEQ ID NO:21;and a nucleic acid sequence that is at least 95% homologous to SEQ IDNO:23.
 4. The composition of claim 2, wherein at least one nucleotidesequence encoding at least one HPV E6-E7 fusion antigen is selected fromthe group consisting of: nucleotide sequence that encodes SEQ ID NO:2;nucleotide sequence that encodes SEQ ID NO:4; nucleotide sequence thatencodes SEQ ID NO:6; nucleotide sequence that encodes SEQ ID NO:8;nucleotide sequence that encodes SEQ ID NO:20; nucleotide sequence thatencodes SEQ ID NO:22; nucleotide sequence that encodes SEQ ID NO:24; anucleic acid sequence that is at least 95% homologous to a nucleic acidsequence nucleotide sequence that encodes SEQ ID NO:2; a nucleic acidsequence that is at least 95% homologous to a nucleotide sequence thatencodes SEQ ID NO:4; a nucleic acid sequence that is at least 95%homologous to a nucleotide sequence that encodes SEQ ID NO:6; a nucleicacid sequence that is at least 95% homologous to a nucleotide sequencethat encodes SEQ ID NO:8; a nucleic acid sequence that is at least 95%homologous to a nucleotide sequence that encodes SEQ ID NO:20; a nucleicacid sequence that is at least 95% homologous to a nucleotide sequencethat encodes SEQ ID NO:22; and a nucleic acid sequence that is at least95% homologous to a nucleotide sequence that encodes SEQ ID NO:24. 5.The composition of claim 2, wherein at least one nucleotide sequenceencoding at least one HPV E6-E7 fusion antigen is selected from thegroup consisting of: nucleotide sequence that encodes SEQ ID NO:6;nucleotide sequence that encodes SEQ ID NO:8; nucleotide sequence thatencodes SEQ ID NO:20; nucleotide sequence that encodes SEQ ID NO:22;nucleotide sequence that encodes SEQ ID NO:24; a nucleic acid sequencethat is at least 95% homologous to a nucleotide sequence that encodesSEQ ID NO:6; a nucleic acid sequence that is at least 95% homologous toa nucleotide sequence that encodes SEQ ID NO:8; a nucleic acid sequencethat is at least 95% homologous to a nucleotide sequence that encodesSEQ ID NO:20; a nucleic acid sequence that is at least 95% homologous toa nucleotide sequence that encodes SEQ ID NO:22; and a nucleic acidsequence that is at least 95% homologous to a nucleotide sequence thatencodes SEQ ID NO:24.
 6. The composition of claim 2, wherein one or morenucleotide sequences encoding the HPV E6-E7 fusion antigen is selectedfrom the group consisting of: nucleotide sequence that encodes SEQ IDNO:2; nucleotide sequence that encodes SEQ ID NO:4; nucleotide sequencethat encodes SEQ ID NO:22; nucleotide sequence that encodes SEQ IDNO:24; and a nucleic acid sequence that is at least 95% homologous to anucleic acid sequence that encodes SEQ ID NO:2; a nucleic acid sequencethat is at least 95% homologous to a nucleic acid sequence that encodesSEQ ID NO:4; a nucleic acid sequence that is at least 95% homologous toa nucleic acid sequence that encodes SEQ ID NO:22; and a nucleic acidsequence that is at least 95% homologous to a nucleic acid sequence thatencodes SEQ ID NO:24.
 7. The composition of claim 2, wherein at leastone nucleotide sequence encoding at least one HPV E6-E7 fusion antigenis selected from the group consisting of: nucleotide sequence thatencodes SEQ ID NO:20; nucleotide sequence that encodes SEQ ID NO:22; anucleic acid sequence that is at least 95% homologous to a nucleotidesequence that encodes SEQ ID NO:20; and a nucleic acid sequence that isat least 95% homologous to a nucleotide sequence that encodes SEQ IDNO:22.
 8. The composition of claim 2, wherein at least one nucleotidesequence encoding at least one HPV E6-E7 fusion antigen has at least 98%homology with at least one nucleotide sequence selected from the groupconsisting of: nucleotide sequence that encodes SEQ ID NO:2; nucleotidesequence that encodes SEQ ID NO:4; nucleotide sequence that encodes SEQID NO:6; nucleotide sequence that encodes SEQ ID NO:8; nucleotidesequence that encodes SEQ ID NO:20; nucleotide sequence that encodes SEQID NO:22; and nucleotide sequence that encodes SEQ ID NO:24.
 9. Thecomposition of claim 2, wherein at least one nucleotide sequenceencoding at least one HPV E6-E7 fusion antigen has at least 99% homologywith at least one nucleotide sequence selected from the group consistingof: nucleotide sequence that encodes SEQ ID NO:2; nucleotide sequencethat encodes SEQ ID NO:4; nucleotide sequence that encodes SEQ ID NO:6;nucleotide sequence that encodes SEQ ID NO:8; nucleotide sequence thatencodes SEQ ID NO:20; nucleotide sequence that encodes SEQ ID NO:22; andnucleotide sequence that encodes SEQ ID NO:24.
 10. The composition ofclaim 2, where the nucleotide sequences encoding the HPV E6-E7 fusionantigen are without a leader sequence at 5′ end that is a nucleotidesequence that encodes SEQ ID NO:10.
 11. The composition of claim 3,comprising one or more nucleotide sequences encoding an HPV E6-E7 fusionantigen selected from the group consisting of: SEQ ID NO:5; SEQ ID NO:7;SEQ ID NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; a nucleic acidsequence that is at least 95% homologous to SEQ ID NO:5; a nucleic acidsequence that is at least 95% homologous to SEQ ID NO:7; a nucleic acidsequence that is at least 95% homologous to SEQ ID NO: 17; a nucleicacid sequence that is at least 95% homologous to SEQ ID NO: 19; anucleic acid sequence that is at least 95% homologous to SEQ ID NO:21;and a nucleic acid sequence that is at least 95% homologous to SEQ IDNO:23.
 12. The composition of claim 7, comprising one or more nucleotidesequences encoding an HPV E6-E7 fusion antigen selected from the groupconsisting of: SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:21; SEQ ID NO:23; anucleic acid sequence that is at least 95% homologous to SEQ ID NO:1; anucleic acid sequence that is at least 95% homologous to SEQ ID NO:3; anucleic acid sequence that is at least 95% homologous to SEQ ID NO:21;and a nucleic acid sequence that is at least 95% homologous to SEQ IDNO:23.
 13. The composition of claim 3, comprising one or more nucleotidesequences encoding an HPV E6-E7 fusion antigen selected from the groupconsisting of: SEQ ID NO:17; SEQ ID NO:19; SEQ ID NO:21; a nucleic acidsequence that is at least 95% homologous to SEQ ID NO: 17; a nucleicacid sequence that is at least 95% homologous to SEQ ID NO:19; and anucleic acid sequence that is at least 95% homologous to SEQ ID NO:21.14. The composition of claim 3, wherein one or more nucleotide sequencesencoding the HPV E6-E7 fusion antigen has at least 98% homology withnucleotide sequences selected from the group consisting of: SEQ ID NO:1;SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:17; SEQ ID NO:19; SEQID NO:21; and SEQ ID NO:
 23. 15. The composition of claim 3, wherein oneor more nucleotide sequences encoding the HPV E6-E7 fusion antigen hasat least 99% homology with nucleotide sequences selected from the groupconsisting of: SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQID NO:17; SEQ ID NO:19; SEQ ID NO:21; and SEQ ID NO:
 23. 16. Thecomposition of claim 7, where the nucleotide sequences encoding the HPVE6-E7 fusion antigen are without a leader sequence at 5′ end that hasnucleotide sequence SEQ ID NO:9.
 17. The composition of claim 2, whereinsaid nucleotide sequence is a plasmid.
 18. A pharmaceutical compositioncomprising a nucleotide sequence of claim
 2. 19. A method of inducing aneffective immune response in an individual against more than one subtypeof HPV, comprising administering to said individual a compositioncomprising a nucleotide sequence of claim
 2. 20. The method of claim 19,wherein said nucleotide sequence is administered into the individual byelectroporation.
 21. A method of inducing an effective immune responsein an individual against more than one subtype of HPV, comprisingadministering to said individual a composition comprising a nucleotidesequence of claim
 3. 22. The method of claim 21, wherein said nucleotidesequence is administered into the individual by electroporation.